P52rIPK Regulates the Molecular Cochaperone P58IPK To Mediate Control of the RNA-Dependent Protein Kinase in Response to Cytoplasmic Stress†
Identifieur interne : 002774 ( Istex/Corpus ); précédent : 002773; suivant : 002775P52rIPK Regulates the Molecular Cochaperone P58IPK To Mediate Control of the RNA-Dependent Protein Kinase in Response to Cytoplasmic Stress†
Auteurs : Michael Gale ; Collin M. Blakely ; André Darveau ; Patrick R. Romano ; Marcus J. Korth ; Michael G. KatzeSource :
- Biochemistry [ 0006-2960 ] ; 2002.
Abstract
The 52 kDa protein referred to as P52rIPK was first identified as a regulator of P58IPK, a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52rIPK and P58IPK each possess structural domains implicated in stress signaling, including the charged domain of P52rIPK and the tetratricopeptide repeat (TPR) and DnaJ domains of P58IPK. The P52rIPK charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52rIPK function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58IPK. This domain bound specifically to P58IPK TPR domain 7, the domain adjacent to the TPR motif required for P58IPK interaction with PKR, thus providing a mechanism for P52rIPK inhibition of P58IPK function. Both the charged domain of P52rIPK and the TPR 7 domain of P58IPK were required for P52rIPK to mediate downstream control of PKR activity, eIF2α phosphorylation, and cell growth. Furthermore, we found that P52rIPK and P58IPK formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52rIPK charged domain functions as a TPR-specific signaling motif to directly regulate P58IPK within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.
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DOI: 10.1021/bi020397e
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ISTEX:496B39CB24F6FA7131E3382C0F0D7617C71A8862Le document en format XML
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Korth</name><affiliation><mods:affiliation>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Washington.</mods:affiliation></affiliation></author><author><name sortKey="Katze, Michael G" sort="Katze, Michael G" uniqKey="Katze M" first="Michael G." last="Katze">Michael G. 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E-mail: honey@u.washington.edu.</mods:affiliation></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">ISTEX</idno><idno type="RBID">ISTEX:496B39CB24F6FA7131E3382C0F0D7617C71A8862</idno><date when="2002" year="2002">2002</date><idno type="doi">10.1021/bi020397e</idno><idno type="url">https://api.istex.fr/ark:/67375/TPS-5VJNB1S2-X/fulltext.pdf</idno><idno type="wicri:Area/Istex/Corpus">002774</idno><idno type="wicri:explorRef" wicri:stream="Istex" wicri:step="Corpus" wicri:corpus="ISTEX">002774</idno></publicationStmt><sourceDesc><biblStruct><analytic><title level="a" type="main">P52<hi rend="superscript">rIPK</hi> Regulates the Molecular Cochaperone P58<hi rend="superscript">IPK</hi> To Mediate Control of the
RNA-Dependent Protein Kinase in Response to Cytoplasmic Stress<ref type="bib" target="#bi020397eAF2"><hi rend="superscript">†</hi></ref></title><author><name sortKey="Gale, Michael" sort="Gale, Michael" uniqKey="Gale M" first="Michael" last="Gale">Michael Gale</name><affiliation><mods:affiliation>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Texas Southwestern Medical Center.</mods:affiliation></affiliation></author><author><name sortKey="Blakely, Collin M" sort="Blakely, Collin M" uniqKey="Blakely C" first="Collin M." last="Blakely">Collin M. Blakely</name><affiliation><mods:affiliation>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Washington.</mods:affiliation></affiliation><affiliation><mods:affiliation> Current address: University of Pennsylvania School of Medicine,Philadelphia, PA.</mods:affiliation></affiliation></author><author><name sortKey="Darveau, Andre" sort="Darveau, Andre" uniqKey="Darveau A" first="André" last="Darveau">André Darveau</name><affiliation><mods:affiliation>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</mods:affiliation></affiliation><affiliation><mods:affiliation> Université Laval.</mods:affiliation></affiliation></author><author><name sortKey="Romano, Patrick R" sort="Romano, Patrick R" uniqKey="Romano P" first="Patrick R." last="Romano">Patrick R. Romano</name><affiliation><mods:affiliation>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</mods:affiliation></affiliation><affiliation><mods:affiliation> Thomas Jefferson University.</mods:affiliation></affiliation></author><author><name sortKey="Korth, Marcus J" sort="Korth, Marcus J" uniqKey="Korth M" first="Marcus J." last="Korth">Marcus J. Korth</name><affiliation><mods:affiliation>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Washington.</mods:affiliation></affiliation></author><author><name sortKey="Katze, Michael G" sort="Katze, Michael G" uniqKey="Katze M" first="Michael G." last="Katze">Michael G. Katze</name><affiliation><mods:affiliation>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</mods:affiliation></affiliation><affiliation><mods:affiliation> University of Washington.</mods:affiliation></affiliation><affiliation><mods:affiliation> To whom correspondence should be addressed. Tel: 206-732-6136.Fax: 206-732-6056. E-mail: honey@u.washington.edu.</mods:affiliation></affiliation></author></analytic><monogr></monogr><series><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="ISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2002</date><date type="published">2002</date><biblScope unit="vol">41</biblScope><biblScope unit="issue">39</biblScope><biblScope unit="page" from="11878">11878</biblScope><biblScope unit="page" to="11887">11887</biblScope></imprint><idno type="ISSN">0006-2960</idno></series></biblStruct></sourceDesc><seriesStmt><idno type="ISSN">0006-2960</idno></seriesStmt></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract">The 52 kDa protein referred to as P52rIPK was first identified as a regulator of P58IPK, a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52rIPK and P58IPK each possess structural domains implicated in stress signaling, including the charged domain of P52rIPK and the tetratricopeptide repeat (TPR) and DnaJ domains of P58IPK. The P52rIPK charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52rIPK function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58IPK. This domain bound specifically to P58IPK TPR domain 7, the domain adjacent to the TPR motif required for P58IPK interaction with PKR, thus providing a mechanism for P52rIPK inhibition of P58IPK function. Both the charged domain of P52rIPK and the TPR 7 domain of P58IPK were required for P52rIPK to mediate downstream control of PKR activity, eIF2α phosphorylation, and cell growth. Furthermore, we found that P52rIPK and P58IPK formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52rIPK charged domain functions as a TPR-specific signaling motif to directly regulate P58IPK within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.</div></front></TEI><istex><corpusName>acs</corpusName><keywords><teeft><json:string>kinase</json:string><json:string>eif2r</json:string><json:string>translational</json:string><json:string>phosphorylation</json:string><json:string>protein kinase</json:string><json:string>biol</json:string><json:string>katze</json:string><json:string>deletion</json:string><json:string>translational control</json:string><json:string>tetratricopeptide</json:string><json:string>gal4</json:string><json:string>mutant</json:string><json:string>gcn2</json:string><json:string>galactose</json:string><json:string>biochemistry</json:string><json:string>eif2r phosphorylation</json:string><json:string>coexpressing</json:string><json:string>fusion 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Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</json:string><json:string>University of Texas Southwestern Medical Center.</json:string></affiliations></json:item><json:item><name>BLAKELY Collin M.</name><affiliations><json:string>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</json:string><json:string>University of Washington.</json:string><json:string>Current address: University of Pennsylvania School of Medicine,Philadelphia, PA.</json:string></affiliations></json:item><json:item><name>DARVEAU André</name><affiliations><json:string>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</json:string><json:string>Université Laval.</json:string></affiliations></json:item><json:item><name>ROMANO Patrick R.</name><affiliations><json:string>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</json:string><json:string>Thomas Jefferson University.</json:string></affiliations></json:item><json:item><name>KORTH Marcus J.</name><affiliations><json:string>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</json:string><json:string>University of Washington.</json:string></affiliations></json:item><json:item><name>KATZE Michael G.</name><affiliations><json:string>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and theJefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901</json:string><json:string>University of Washington.</json:string><json:string>To whom correspondence should be addressed. Tel: 206-732-6136.Fax: 206-732-6056. E-mail: honey@u.washington.edu.</json:string></affiliations></json:item></author><arkIstex>ark:/67375/TPS-5VJNB1S2-X</arkIstex><language><json:string>eng</json:string></language><originalGenre><json:string>research-article</json:string></originalGenre><abstract>The 52 kDa protein referred to as P52rIPK was first identified as a regulator of P58IPK, a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52rIPK and P58IPK each possess structural domains implicated in stress signaling, including the charged domain of P52rIPK and the tetratricopeptide repeat (TPR) and DnaJ domains of P58IPK. The P52rIPK charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52rIPK function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58IPK. This domain bound specifically to P58IPK TPR domain 7, the domain adjacent to the TPR motif required for P58IPK interaction with PKR, thus providing a mechanism for P52rIPK inhibition of P58IPK function. Both the charged domain of P52rIPK and the TPR 7 domain of P58IPK were required for P52rIPK to mediate downstream control of PKR activity, eIF2α phosphorylation, and cell growth. Furthermore, we found that P52rIPK and P58IPK formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52rIPK charged domain functions as a TPR-specific signaling motif to directly regulate P58IPK within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.</abstract><qualityIndicators><score>9.664</score><pdfWordCount>8512</pdfWordCount><pdfCharCount>53252</pdfCharCount><pdfVersion>1.2</pdfVersion><pdfPageCount>10</pdfPageCount><pdfPageSize>612 x 792 pts (letter)</pdfPageSize><pdfWordsPerPage>851</pdfWordsPerPage><pdfText>true</pdfText><refBibsNative>true</refBibsNative><abstractWordCount>222</abstractWordCount><abstractCharCount>1462</abstractCharCount><keywordCount>0</keywordCount></qualityIndicators><title>P52rIPK Regulates the Molecular Cochaperone P58IPK To Mediate Control of the RNA-Dependent Protein Kinase in Response to Cytoplasmic Stress†</title><genre><json:string>research-article</json:string></genre><host><title>Biochemistry</title><language><json:string>unknown</json:string></language><issn><json:string>0006-2960</json:string></issn><eissn><json:string>1520-4995</json:string></eissn><volume>41</volume><issue>39</issue><pages><first>11878</first><last>11887</last></pages><genre><json:string>journal</json:string></genre></host><ark><json:string>ark:/67375/TPS-5VJNB1S2-X</json:string></ark><categories><wos><json:string>1 - science</json:string><json:string>2 - biochemistry & molecular biology</json:string></wos><scienceMetrix><json:string>1 - health sciences</json:string><json:string>2 - biomedical research</json:string><json:string>3 - biochemistry & molecular biology</json:string></scienceMetrix><scopus><json:string>1 - Life Sciences</json:string><json:string>2 - Biochemistry, Genetics and Molecular Biology</json:string><json:string>3 - Biochemistry</json:string></scopus><inist><json:string>1 - sciences appliquees, technologies et medecines</json:string><json:string>2 - sciences biologiques et medicales</json:string><json:string>3 - sciences biologiques fondamentales et appliquees. psychologie</json:string></inist></categories><publicationDate>2002</publicationDate><copyrightDate>2002</copyrightDate><doi><json:string>10.1021/bi020397e</json:string></doi><id>496B39CB24F6FA7131E3382C0F0D7617C71A8862</id><score>1</score><fulltext><json:item><extension>pdf</extension><original>true</original><mimetype>application/pdf</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-5VJNB1S2-X/fulltext.pdf</uri></json:item><json:item><extension>zip</extension><original>false</original><mimetype>application/zip</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-5VJNB1S2-X/bundle.zip</uri></json:item><json:item><extension>txt</extension><original>false</original><mimetype>text/plain</mimetype><uri>https://api.istex.fr/ark:/67375/TPS-5VJNB1S2-X/fulltext.txt</uri></json:item><istex:fulltextTEI uri="https://api.istex.fr/ark:/67375/TPS-5VJNB1S2-X/fulltext.tei"><teiHeader><fileDesc><titleStmt><title level="a" type="main">P52<hi rend="superscript">rIPK</hi> Regulates the Molecular Cochaperone P58<hi rend="superscript">IPK</hi> To Mediate Control of the
RNA-Dependent Protein Kinase in Response to Cytoplasmic Stress<ref type="bib" target="#bi020397eAF2"><hi rend="superscript">†</hi></ref></title></titleStmt><publicationStmt><authority>ISTEX</authority><publisher>American Chemical Society</publisher><availability><licence>Copyright © 2002 American Chemical Society</licence><p>American Chemical Society</p></availability><date type="published">2002</date><date type="Copyright" when="2002">2002</date></publicationStmt><notesStmt><note type="content-type" source="research-article" scheme="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</note><note type="publication-type" scheme="https://publication-type.data.istex.fr/ark:/67375/JMC-0GLKJH51-B">journal</note></notesStmt><sourceDesc><biblStruct type="article"><analytic><title level="a" type="main">P52<hi rend="superscript">rIPK</hi> Regulates the Molecular Cochaperone P58<hi rend="superscript">IPK</hi> To Mediate Control of the
RNA-Dependent Protein Kinase in Response to Cytoplasmic Stress<ref type="bib" target="#bi020397eAF2"><hi rend="superscript">†</hi></ref></title><author xml:id="author-0000"><persName><surname>Gale,</surname><forename type="first">Michael</forename></persName><affiliation><orgName type="institution">Department of Microbiology and The Simmons Comprehensive Cancer Center</orgName><orgName type="institution">University of Texas Southwestern Medical Center</orgName><address><addrLine>Dallas</addrLine><addrLine>Texas 75390</addrLine><addrLine>Department of Microbiology</addrLine><addrLine>University of Washington</addrLine><addrLine>Seattle</addrLine><addrLine>Washington 98195</addrLine><addrLine>Département de Biochimie et Microbiologie</addrLine><addrLine>Université Laval</addrLine><addrLine>Québec</addrLine><country key="CA" xml:lang="en">CANADA</country><addrLine>and Thomas Jefferson University and the Jefferson Center for Biomedical Research</addrLine><addrLine>Doylestown</addrLine><addrLine>Pennsylvania 18901</addrLine></address></affiliation><note place="foot" n="bi020397eAF3"><ref>‡</ref><p>
University of Texas Southwestern Medical Center.</p></note></author><author xml:id="author-0001"><persName><surname>Blakely</surname><forename type="first">Collin M.</forename></persName><note place="foot" n="bi020397eAF4"><ref>§</ref><p>
University of Washington.</p></note><note place="foot" n="bi020397eAF5"><ref>‖</ref><p>
Current address: University of Pennsylvania School of Medicine,
Philadelphia, PA.</p></note></author><author xml:id="author-0002"><persName><surname>Darveau</surname><forename type="first">André</forename></persName><affiliation><orgName type="institution">Department of Microbiology and The Simmons Comprehensive Cancer Center</orgName><orgName type="institution">University of Texas Southwestern Medical Center</orgName><address><addrLine>Dallas</addrLine><addrLine>Texas 75390</addrLine><addrLine>Department of Microbiology</addrLine><addrLine>University of Washington</addrLine><addrLine>Seattle</addrLine><addrLine>Washington 98195</addrLine><addrLine>Département de Biochimie et Microbiologie</addrLine><addrLine>Université Laval</addrLine><addrLine>Québec</addrLine><country key="CA" xml:lang="en">CANADA</country><addrLine>and Thomas Jefferson University and the Jefferson Center for Biomedical Research</addrLine><addrLine>Doylestown</addrLine><addrLine>Pennsylvania 18901</addrLine></address></affiliation><note place="foot" n="bi020397eAF6"><ref>⊥</ref><p>
Université Laval.</p></note></author><author xml:id="author-0003"><persName><surname>Romano</surname><forename type="first">Patrick R.</forename></persName><affiliation><orgName type="institution">Department of Microbiology and The Simmons Comprehensive Cancer Center</orgName><orgName type="institution">University of Texas Southwestern Medical Center</orgName><address><addrLine>Dallas</addrLine><addrLine>Texas 75390</addrLine><addrLine>Department of Microbiology</addrLine><addrLine>University of Washington</addrLine><addrLine>Seattle</addrLine><addrLine>Washington 98195</addrLine><addrLine>Département de Biochimie et Microbiologie</addrLine><addrLine>Université Laval</addrLine><addrLine>Québec</addrLine><country key="CA" xml:lang="en">CANADA</country><addrLine>and Thomas Jefferson University and the Jefferson Center for Biomedical Research</addrLine><addrLine>Doylestown</addrLine><addrLine>Pennsylvania 18901</addrLine></address></affiliation><note place="foot" n="bi020397eAF10"><ref>#</ref><p>
Thomas Jefferson University.</p></note></author><author xml:id="author-0004"><persName><surname>Korth</surname><forename type="first">Marcus J.</forename></persName><affiliation><orgName type="institution">Department of Microbiology and The Simmons Comprehensive Cancer Center</orgName><orgName type="institution">University of Texas Southwestern Medical Center</orgName><address><addrLine>Dallas</addrLine><addrLine>Texas 75390</addrLine><addrLine>Department of Microbiology</addrLine><addrLine>University of Washington</addrLine><addrLine>Seattle</addrLine><addrLine>Washington 98195</addrLine><addrLine>Département de Biochimie et Microbiologie</addrLine><addrLine>Université Laval</addrLine><addrLine>Québec</addrLine><country key="CA" xml:lang="en">CANADA</country><addrLine>and Thomas Jefferson University and the Jefferson Center for Biomedical Research</addrLine><addrLine>Doylestown</addrLine><addrLine>Pennsylvania 18901</addrLine></address></affiliation><note place="foot" n="bi020397eAF4"><ref>§</ref><p>
University of Washington.</p></note></author><author xml:id="author-0005" role="corresp"><persName><surname>Katze</surname><forename type="first">Michael G.</forename></persName><note place="foot" n="bi020397eAF4"><ref>§</ref><p>
University of Washington.</p></note><affiliation role="corresp"> To whom correspondence should be addressed. Tel: 206-732-6136. Fax: 206-732-6056. E-mail: honey@u.washington.edu.</affiliation></author><idno type="istex">496B39CB24F6FA7131E3382C0F0D7617C71A8862</idno><idno type="ark">ark:/67375/TPS-5VJNB1S2-X</idno><idno type="DOI">10.1021/bi020397e</idno></analytic><monogr><title level="j" type="main">Biochemistry</title><title level="j" type="abbrev">Biochemistry</title><idno type="acspubs">bi</idno><idno type="coden">bichaw</idno><idno type="pISSN">0006-2960</idno><idno type="eISSN">1520-4995</idno><imprint><publisher>American Chemical Society</publisher><date type="e-published">2002</date><date type="published">2002</date><biblScope unit="vol">41</biblScope><biblScope unit="issue">39</biblScope><biblScope unit="page" from="11878">11878</biblScope><biblScope unit="page" to="11887">11887</biblScope></imprint></monogr></biblStruct></sourceDesc></fileDesc><encodingDesc><schemaRef type="ODD" url="https://xml-schema.delivery.istex.fr/tei-istex.odd"></schemaRef><appInfo><application ident="pub2tei" version="1.0.41" when="2020-04-06"><label>pub2TEI-ISTEX</label><desc>A set of style sheets for converting XML documents encoded in various scientific publisher formats into a common TEI format.
<ref target="http://www.tei-c.org/">We use TEI</ref></desc></application></appInfo></encodingDesc><profileDesc><abstract><p>The 52 kDa protein referred to as P52<hi rend="superscript">rIPK</hi> was first identified as a regulator of P58<hi rend="superscript">IPK</hi>, a cellular
inhibitor of the RNA-dependent protein kinase (PKR). P52<hi rend="superscript">rIPK</hi> and P58<hi rend="superscript">IPK</hi> each possess structural domains
implicated in stress signaling, including the charged domain of P52<hi rend="superscript">rIPK</hi> and the tetratricopeptide repeat
(TPR) and DnaJ domains of P58<hi rend="superscript">IPK</hi>. The P52<hi rend="superscript">rIPK</hi> charged domain exhibits homology to the charged domains
of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of
P52<hi rend="superscript">rIPK</hi> function and expression, which first revealed that the 114 amino acid charged domain was necessary
and sufficient for interaction with P58<hi rend="superscript">IPK</hi>. This domain bound specifically to P58<hi rend="superscript">IPK</hi> TPR domain 7, the
domain adjacent to the TPR motif required for P58<hi rend="superscript">IPK</hi> interaction with PKR, thus providing a mechanism
for P52<hi rend="superscript">rIPK</hi> inhibition of P58<hi rend="superscript">IPK</hi> function. Both the charged domain of P52<hi rend="superscript">rIPK</hi> and the TPR 7 domain of
P58<hi rend="superscript">IPK</hi> were required for P52<hi rend="superscript">rIPK</hi> to mediate downstream control of PKR activity, eIF2α phosphorylation,
and cell growth. Furthermore, we found that P52<hi rend="superscript">rIPK</hi> and P58<hi rend="superscript">IPK</hi> formed a stable intracellular complex
during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in
which the P52<hi rend="superscript">rIPK</hi> charged domain functions as a TPR-specific signaling motif to directly regulate P58<hi rend="superscript">IPK</hi>
within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular
mRNA translation.
</p></abstract><textClass ana="subject"><keywords scheme="document-type-name"><term>Article</term></keywords></textClass><langUsage><language ident="en"></language></langUsage></profileDesc><revisionDesc><change when="2020-04-06" who="#istex" xml:id="pub2tei">formatting</change></revisionDesc></teiHeader></istex:fulltextTEI></fulltext><metadata><istex:metadataXml wicri:clean="corpus acs not found" wicri:toSee="no header"><istex:xmlDeclaration>version="1.0" encoding="UTF-8"</istex:xmlDeclaration><istex:document><article article-type="research-article" specific-use="acs2jats-1.1.23" dtd-version="1.1d1"><front><journal-meta><journal-id journal-id-type="acspubs">bi</journal-id><journal-id journal-id-type="coden">bichaw</journal-id><journal-title-group><journal-title>Biochemistry</journal-title><abbrev-journal-title>Biochemistry</abbrev-journal-title></journal-title-group><issn pub-type="ppub">0006-2960</issn><issn pub-type="epub">1520-4995</issn><publisher><publisher-name>American Chemical Society</publisher-name></publisher><self-uri>pubs.acs.org/biochemistry</self-uri></journal-meta><article-meta><article-id pub-id-type="doi">10.1021/bi020397e</article-id><article-categories><subj-group subj-group-type="document-type-name"><subject>Article</subject></subj-group></article-categories><title-group><article-title>P52<sup>rIPK</sup> Regulates the Molecular Cochaperone P58<sup>IPK</sup> To Mediate Control of the
RNA-Dependent Protein Kinase in Response to Cytoplasmic Stress<xref rid="bi020397eAF2"><sup>†</sup></xref></article-title></title-group><contrib-group><contrib contrib-type="author"><name name-style="western"><surname>Gale,</surname><given-names>Michael</given-names></name><xref rid="bi020397eAF3"><sup>‡</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Blakely</surname><given-names>Collin M.</given-names></name><xref rid="bi020397eAF4"><sup>§</sup></xref><xref rid="bi020397eAF5"><sup>‖</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Darveau</surname><given-names>André</given-names></name><xref rid="bi020397eAF6"><sup>⊥</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Romano</surname><given-names>Patrick R.</given-names></name><xref rid="bi020397eAF10"><sup>#</sup></xref></contrib><contrib contrib-type="author"><name name-style="western"><surname>Korth</surname><given-names>Marcus J.</given-names></name><xref rid="bi020397eAF4"><sup>§</sup></xref></contrib><contrib contrib-type="author" corresp="yes"><name name-style="western"><surname>Katze</surname><given-names>Michael G.</given-names></name><xref rid="bi020397eAF1">*</xref><xref rid="bi020397eAF4"><sup>§</sup></xref></contrib><aff>Department of Microbiology and The Simmons Comprehensive Cancer Center, University of Texas Southwestern
Medical Center, Dallas, Texas 75390, Department of Microbiology, University of Washington, Seattle, Washington 98195,
Département de Biochimie et Microbiologie, Université Laval, Québec, Canada, and Thomas Jefferson University and the
Jefferson Center for Biomedical Research, Doylestown, Pennsylvania 18901
</aff></contrib-group><author-notes><fn id="bi020397eAF3"><label>‡</label><p>
University of Texas Southwestern Medical Center.</p></fn><fn id="bi020397eAF4"><label>§</label><p>
University of Washington.</p></fn><fn id="bi020397eAF5"><label>‖</label><p>
Current address: University of Pennsylvania School of Medicine,
Philadelphia, PA.</p></fn><fn id="bi020397eAF6"><label>⊥</label><p>
Université Laval.</p></fn><fn id="bi020397eAF10"><label>#</label><p>
Thomas Jefferson University.</p></fn><corresp id="bi020397eAF1">
To whom correspondence should be addressed. Tel: 206-732-6136.
Fax: 206-732-6056. E-mail: honey@u.washington.edu.</corresp></author-notes><pub-date pub-type="epub"><day>20</day><month>08</month><year>2002</year></pub-date><pub-date pub-type="ppub"><day>01</day><month>10</month><year>2002</year></pub-date><volume>41</volume><issue>39</issue><fpage>11878</fpage><lpage>11887</lpage><history><date date-type="received"><day>03</day><month>06</month><year>2002</year></date><date date-type="rev-recd"><day>19</day><month>07</month><year>2002</year></date><date date-type="asap"><day>20</day><month>08</month><year>2002</year></date><date date-type="issue-pub"><day>01</day><month>10</month><year>2002</year></date></history><permissions><copyright-statement>Copyright © 2002 American Chemical Society</copyright-statement><copyright-year>2002</copyright-year><copyright-holder>American Chemical Society</copyright-holder></permissions><abstract><p>The 52 kDa protein referred to as P52<sup>rIPK</sup> was first identified as a regulator of P58<sup>IPK</sup>, a cellular
inhibitor of the RNA-dependent protein kinase (PKR). P52<sup>rIPK</sup> and P58<sup>IPK</sup> each possess structural domains
implicated in stress signaling, including the charged domain of P52<sup>rIPK</sup> and the tetratricopeptide repeat
(TPR) and DnaJ domains of P58<sup>IPK</sup>. The P52<sup>rIPK</sup> charged domain exhibits homology to the charged domains
of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of
P52<sup>rIPK</sup> function and expression, which first revealed that the 114 amino acid charged domain was necessary
and sufficient for interaction with P58<sup>IPK</sup>. This domain bound specifically to P58<sup>IPK</sup> TPR domain 7, the
domain adjacent to the TPR motif required for P58<sup>IPK</sup> interaction with PKR, thus providing a mechanism
for P52<sup>rIPK</sup> inhibition of P58<sup>IPK</sup> function. Both the charged domain of P52<sup>rIPK</sup> and the TPR 7 domain of
P58<sup>IPK</sup> were required for P52<sup>rIPK</sup> to mediate downstream control of PKR activity, eIF2α phosphorylation,
and cell growth. Furthermore, we found that P52<sup>rIPK</sup> and P58<sup>IPK</sup> formed a stable intracellular complex
during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in
which the P52<sup>rIPK</sup> charged domain functions as a TPR-specific signaling motif to directly regulate P58<sup>IPK</sup>
within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular
mRNA translation.
</p></abstract><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>bi020397e</meta-value></custom-meta></custom-meta-group></article-meta><notes id="bi020397eAF2"><label>†</label><p>
Financial support was provided by National Institutes of Health
Grants AI22646 (to M.G.K.) and AI48235 (to M.G.). M.G. also received
support from the Helen Hay Whitney Foundation and the State of Texas
Applied Research Program (no. 0138-1999).</p></notes></front><body><sec id="d7e251"><title></title><p>Stress-induced translational control pathways provide the
cell with a rapid mechanism by which to alter gene
expression in response to a wide spectrum of signals,
including heat and chemical stressors, osmotic shock,
ultraviolet radiation, nutrient deprivation, and virus infection
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00001" ref-type="bibr"></xref>−<xref rid="bi020397eb00002" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi020397eb00003" ref-type="bibr"></xref></named-content></italic>). A key feature of these pathways is the reversible
phosphorylation of translation factors, which enforces translational control programs by modifying translation factor
function (<italic toggle="yes"><xref rid="bi020397eb00004" ref-type="bibr"></xref></italic>). The major points of translational control reside
within the processes of translation initiation. In this often
rate-limiting step, the initiator Met-tRNA<sub>i</sub> is delivered to the
40S ribosome preinitiation complex by eukaryotic initiation
factor 2 (eIF2)<xref rid="bi020397eb00001" ref-type="bibr"></xref> (<italic toggle="yes"><xref rid="bi020397eb00005" ref-type="bibr"></xref></italic>). In mammalian cells, eIF2 function is
regulated through phosphorylation of its α subunit (eIF2α)
by the actions of a family of eIF2α protein kinases that
includes HRI, GCN2, PERK/PEK, and PKR (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00006" ref-type="bibr"></xref>, <xref rid="bi020397eb00007" ref-type="bibr"></xref></named-content></italic>). Phosphorylation of eIF2α blocks a critical guanine nucleotide
exchange reaction, leaving eIF2 in an inactive GDP-bound
form that is unavailable for initiation of mRNA translation
(<italic toggle="yes"><xref rid="bi020397eb00008" ref-type="bibr"></xref></italic>).
</p><p>The eIF2α protein kinases possess common structural
features within their catalytic domains that direct substrate
specificity for eIF2α (<italic toggle="yes"><xref rid="bi020397eb00007" ref-type="bibr"></xref></italic>). However, these kinases exhibit
significant sequence divergence within their regulatory
domains, which likely contributes to the differing array of
signals to which each kinase responds. In erythroid cells,
HRI regulates protein synthesis in response to hemin levels
and is activated by heme deprivation (<italic toggle="yes"><xref rid="bi020397eb00009" ref-type="bibr"></xref></italic>). In <italic toggle="yes">Saccharomyces
cerevisiae,</italic> GCN2 is activated in response to nutrient
deprivation that leads to amino acid (aa) limitation. By
phosphorylating eIF2α, GCN2 stimulates the translation of
<italic toggle="yes">GCN4</italic> mRNA to induce aa biosynthesis (<italic toggle="yes"><xref rid="bi020397eb00010" ref-type="bibr"></xref></italic>). GCN2 homologues have also been identified in mammalian and insect
tissues (<italic toggle="yes">8</italic>, <italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00011" ref-type="bibr"></xref>−<xref rid="bi020397eb00012" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi020397eb00013" ref-type="bibr"></xref></named-content></italic>; however, the extent to which the
metazoan GCN2 homologues regulate aa biosynthesis, and
their role in translational signaling, remains to be determined.
The PERK/PEK eIF2α kinase is localized to the endoplasmic
reticulum (ER) membrane and is activated in response to
ER stress (<italic toggle="yes"><xref rid="bi020397eb00014" ref-type="bibr"></xref></italic>). PERK/PEK participates in the unfolded
protein response by phosphorylating eIF2α to limit local
mRNA translation (<italic toggle="yes"><xref rid="bi020397eb00015" ref-type="bibr"></xref></italic>), which is thought to relieve ER stress
by reducing the de novo synthesis of proteins destined for
the ER.
</p><p>PKR is a cytoplasmic protein present in most mammalian
tissues and is activated upon binding to double-stranded RNA
(dsRNA) or by interacting with specific protein activators
(<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00016" ref-type="bibr"></xref>, <xref rid="bi020397eb00017" ref-type="bibr"></xref></named-content></italic>). PKR levels increase approximately 3−10-fold in
response to interferons (IFNs), whereupon the kinase plays
a prominent role in mediating the antiviral and antiproliferative actions of these cytokines (<italic toggle="yes"><xref rid="bi020397eb00018" ref-type="bibr"></xref></italic>). PKR also mediates
the response to dsRNA signals that stimulate apoptosis and
that activate NFκB and IRF-1 (<italic toggle="yes"><xref rid="bi020397eb00019" ref-type="bibr"></xref></italic>). During virus infection,
activation of PKR by dsRNA leads to a suppression of
mRNA translation that serves to limit virus replication (<italic toggle="yes"><xref rid="bi020397eb00020" ref-type="bibr"></xref></italic>).
Many viruses have therefore evolved mechanisms to inhibit
PKR (<italic toggle="yes"><xref rid="bi020397eb00018" ref-type="bibr"></xref></italic>), including influenza virus, which activates P58<sup>IPK</sup>,
a cellular PKR inhibitor (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00021" ref-type="bibr"></xref>−<xref rid="bi020397eb00022" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi020397eb00023" ref-type="bibr"></xref></named-content></italic>).
</p><p>P58<sup>IPK</sup> is a member of the tetratricopeptide repeat (TPR)
domain protein family and encodes nine tandemly arranged
TPR domains. Like other TPR proteins, the TPR domains
of P58<sup>IPK</sup> are important for directing protein interactions,
which in the case of P58<sup>IPK</sup> includes self-association. P58<sup>IPK</sup>
binds to PKR through TPR domain 6 (<italic toggle="yes"><xref rid="bi020397eb00024" ref-type="bibr"></xref></italic>) and inhibits PKR
by disrupting the process of kinase dimerization that is
required for catalytic function (<italic toggle="yes"><xref rid="bi020397eb00022" ref-type="bibr"></xref></italic>). In addition, P58<sup>IPK</sup>
possesses a C-terminal DnaJ domain that formally classifies
it as a member of the stress-response protein superfamily
(<italic toggle="yes">21</italic>,<italic toggle="yes"> 25</italic>,<italic toggle="yes"> 26</italic>). We have also shown that P58<sup>IPK</sup> undergoes
regulatory interactions with heat shock protein (Hsp) 40 that
result in stimulation of Hsp70 function during the heat shock
response (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00023" ref-type="bibr"></xref>, <xref rid="bi020397eb00027" ref-type="bibr"></xref></named-content></italic>). These results define P58<sup>IPK</sup> as a protein
cochaperone that inhibits PKR, possibly by directing Hsp-dependent protein refolding or kinase denaturation. The
inhibition of PKR by P58<sup>IPK</sup> can stimulate cell growth by
disrupting PKR-dependent control of mRNA translation and
by blocking PKR-dependent apoptosis (<italic toggle="yes">25</italic>,<italic toggle="yes"> 28</italic>,<italic toggle="yes"> 29</italic>).
</p><p>P58<sup>IPK</sup> is also regulated through interaction with P52<sup>rIPK</sup>,
a protein with limited homology to the charged domains of
Hsp90 (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>). We found that P52<sup>rIPK</sup> binds to P58<sup>IPK</sup> in vivo
and in vitro and that this interaction blocks the PKR
regulatory function of P58<sup>IPK</sup>. Moreover, when these components are coexpressed in yeast, P52<sup>rIPK</sup> removes the block
to PKR function imposed by P58<sup>IPK</sup>, resulting in restoration
of eIF2α phosphorylation (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>). These studies indicate that
P52<sup>rIPK</sup> functions as an upstream regulator of PKR, eIF2,
and mRNA translation through its ability to interact with
and inhibit P58<sup>IPK</sup>, though the mechanisms of this regulation
have not been defined. In the present report, we describe
the domains of P52<sup>rIPK</sup> and P58<sup>IPK</sup> that are responsible for
the interaction of these proteins and present evidence that
P52<sup>rIPK</sup> functions as an upstream signal transducer to regulate
P58<sup>IPK</sup> in response to cytoplasmic stress.
</p></sec><sec id="d7e467"><title>Experimental Procedures</title><p><italic toggle="yes">Plasmids and Recombinant DNA Construction.</italic> The GAL4
DNA-binding domain (BD) or activation domain (AD) fusion
constructs pBD-P58<sup>IPK</sup>wt, pBD-P58<sup>IPK</sup> ΔTPR6, pBD-P58<sup>IPK</sup>
8−1, pBD-P58<sup>IPK</sup> 8−2, pBD-P58<sup>IPK</sup> 9−1, pAD-PKR K296R,
and pAD-P52<sub>rIPK</sub>wt were described previously (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00024" ref-type="bibr"></xref>, <xref rid="bi020397eb00030" ref-type="bibr"></xref></named-content></italic>).
Plasmids pBD-P58<sup>IPK</sup> ΔTPR5 and pBD-P58<sup>IPK</sup> ΔTPR7 were
constructed by recovering the respective internal <italic toggle="yes">Bst</italic>XI/<italic toggle="yes">Bam</italic>HI fragments from pCDNA1neo-P58<sup>IPK</sup> ΔTPR5 and
ΔTPR7, respectively (<italic toggle="yes"><xref rid="bi020397eb00029" ref-type="bibr"></xref></italic>), and cloning them into <italic toggle="yes">Bst</italic>XI/<italic toggle="yes">Bam</italic>HI-digested pBD-P58<sup>IPK</sup>wt. The pBD-P58<sup>IPK</sup> constructs
8−4, 10−1, and TPR7, encode P58<sup>IPK</sup> aa 1−302, 154−267,
and 268−302, respectively, and were cloned from PCR
products into the <italic toggle="yes">Eco</italic>RI/<italic toggle="yes">Bam</italic>HI sites of pGBT9 to yield in-frame fusion proteins with the GAL4 DNA-binding domain.
The pAD-P52<sup>rIPK</sup> deletion constructs were cloned from PCR
products derived from pAD-P52<sup>rIPK</sup>wt using restriction site-linked oligonucleotide primer pairs. PCR products were first
subcloned into pCR2.1 (Invitrogen). Insert DNA encoding
P52<sup>rIPK</sup> aa 1−243, 237−492, 1−203, 1−84, or 86−203 was
released from pCR2.1 by <italic toggle="yes">Eco</italic>RI/<italic toggle="yes">Bam</italic>HI digestion, and the
gel-purified fragments were cloned into the corresponding
sites of pGAD424 (Clontech) to yield pAD-P52<sup>rIPK</sup> deletion
constructs fused in-frame to the GAL4 activation domain.
DNA encoding P52<sup>rIPK</sup> aa 201−492 was released from
pCR2.1 by <italic toggle="yes">Bam</italic>HI cleavage and subsequently cloned into
<italic toggle="yes">Bam</italic>HI-digested pAD-P52<sup>rIPK</sup> 1−84 to yield pAD-P52<sup>rIPK</sup>
Δ85−200. Plasmids pGex2T-P58<sup>IPK</sup> and pBSK-P52 encode
full-length P58<sup>IPK</sup> and P52<sup>rIPK</sup>, respectively (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>). Inducible
expression of P58<sup>IPK</sup> and P52<sup>rIPK</sup> in yeast cells was achieved
by utilizing the galactose-inducible 2 μm yeast expression
vectors pEMBLyex4 (pYex4, encodes uracil selection) (<italic toggle="yes"><xref rid="bi020397eb00031" ref-type="bibr"></xref></italic>)
and pYX233 (pYX, encodes tryptophan selection) (Novagen),
respectively. Construction of pYex4-P58<sup>IPK</sup>wt and pYX-P52<sup>rIPK</sup>wt was described previously (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>). Plasmids pYX-P52<sup>rIPK</sup> 86−200 and pYX-P52<sup>rIPK</sup> Δ86−200 were constructed
by cloning the <italic toggle="yes">Eco</italic>RI/<italic toggle="yes">Bam</italic>HI fragments from the respective
pAD constructs into the <italic toggle="yes">Eco</italic>RI/<italic toggle="yes">Bam</italic>HI sites of pYX233.
Plasmid pYex4-P58<sup>IPK</sup> ΔTPR7 was derived by cloning the
<italic toggle="yes">Sma</italic>I/<italic toggle="yes">Xba</italic>I fragment from pCDNA1neo- P58<sup>IPK</sup> ΔTPR7 into
<italic toggle="yes">Eco</italic>RV/<italic toggle="yes">Xba</italic>I-digested pYex4. All cloning products were
sequenced by the dye termination method with an Applied
Biosystems automated DNA sequencer to ensure that no
mutations were introduced during PCR amplification or
cloning.
</p><p><italic toggle="yes">Cell Culture.</italic> HeLa, 293, NIH3T3, Cos1 monkey kidney
cells, and Madin Darby bovine kidney cells (MDBK) were
obtained from ATCC. Cells were cultured in Dulbecco's
modified Eagle medium (DMEM) supplemented with 10%
fetal bovine serum (FBS), antibiotics, and 200 μM <sc>l</sc>-glutamine. For stress induction experiments, cultured monolayers (at approximately 60% confluency) were rinsed twice
with sterile PBS and cultured for the indicated time in media
containing 150 μM sodium arsenite (Sigma), 6 mM tunicamycin, or 0.2 mM thapsigargin. For heat shock experiments,
media were removed from HeLa cell monolayers and
replaced with media prewarmed to 42 °C, after which cells
were incubated for a total of 30 min at 42 °C prior to
harvesting. For serum deprivation experiments, HeLa cell
monolayers were rinsed twice with sterile PBS and incubated
for 4 h in media lacking FBS. Control cultures were similarly
rinsed with PBS but were incubated for 4 h in media
containing 10% FBS.
</p><p><italic toggle="yes">Protein Analysis.</italic> Yeast extracts were prepared from 20
mL liquid cultures (<italic toggle="yes"><xref rid="bi020397eb00024" ref-type="bibr"></xref></italic>), and mammalian cell protein extracts
were prepared in lysis buffer as described previously (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>).
Total protein concentration in cell extracts was determined
using the Bio-Rad protein assay. Immunoblot analyses of
proteins separated by SDS−PAGE, or single-dimension
isoelectric focusing, were conducted as described (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>). PKR
or P58<sup>IPK</sup> expression was detected using monoclonal antibody
(Mab) 71/10 (<italic toggle="yes"><xref rid="bi020397eb00032" ref-type="bibr"></xref></italic>) or 9F10, respectively (<italic toggle="yes"><xref rid="bi020397eb00028" ref-type="bibr"></xref></italic>). P52<sup>rIPK</sup> expression was detected using anti-P52<sup>rIPK</sup> polyclonal rabbit serum
(<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>) or the 5D12 Mab described below. BD- and AD-fused
proteins were detected using anti-GAL4 BD and anti-GAL4
AD Mab, respectively (Clontech).
</p><p>For production of 5D12 Mab specific to human P52<sup>rIPK</sup>,
mice were immunized with recombinant GST-P52<sup>rIPK</sup> as
described (<italic toggle="yes"><xref rid="bi020397eb00028" ref-type="bibr"></xref></italic>). Immune serum was screened for reactivity
to GST-P52<sup>rIPK</sup> or GST alone, and mice with specific
reactivity to P52<sup>rIPK</sup> were selected and euthanized, and their
splenocytes were prepared for hybridoma production using
standard procedures (<italic toggle="yes"><xref rid="bi020397eb00033" ref-type="bibr"></xref></italic>). Hybridoma culture supernatants
were screened by immunoblot analysis for the presence of
antibodies specific for recombinant P52<sup>rIPK</sup>. Cells from
positive cultures were cloned by limiting dilution, and the
resulting culture supernatants were rescreened for P52<sup>rIPK</sup>-specific antibodies (IgG<sub>1</sub> subtype).
</p><p>For immunoprecipitation reactions, 1 × 10<sup>5</sup> cpm of [<sup>35</sup>S]methionine-labeled in vitro translation products, or unlabeled
cell extract (400 or 600 μg), was mixed with the indicated
amount of Mab or polyclonal antiserum for 1 h at 4 °C. After
the addition of protein G−agarose beads, mixtures were
incubated for an additional 1 h at 4 °C. Immunocomplexes
were recovered by centrifugation at 12000<italic toggle="yes">g</italic> and washed four
times in lysis buffer (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>) containing an additional 200 mM
NaCl (for a final NaCl concentration of 350 mM). After the
final wash, bead-bound immunocomplexes were released by
the addition of 50 μL of SDS sample buffer, incubated in a
100 °C bath, and placed on ice prior to loading on gels.
Eluted proteins were resolved by 10% or 12.5% SDS−PAGE, and in some experiments, the resolved proteins were
blotted to nitrocellulose membranes and subjected to immunoblot analysis.
</p><p><italic toggle="yes">In Vitro Translation Reactions and GST Pull-Down Assay.</italic>
Full-length and truncated P52<sup>rIPK</sup> constructs were transcribed
from the T7 promoter of pBSK-P52<sup>rIPK</sup> and translated in vitro
in the presence of [<sup>35</sup>S]methionine using the TNT reaction
system (Promega). To generate P52<sup>rIPK</sup> aa 1−384 or 1−239,
we digested pBSK-P52<sup>IPK</sup> with <italic toggle="yes">Mun</italic>I and <italic toggle="yes">Bgl</italic>II restriction
enzymes, respectively, and gel purified the linear plasmids
prior to the TNT reaction. Radiolabel incorporation of in
vitro translated products was quantified by scintillation
counting of trichloroacetic acid-precipitable material. Expression of GST and GST-P58<sup>IPK</sup> in <italic toggle="yes">Escherichia coli</italic> and GST
pull-down assays were carried out as previously described
(<italic toggle="yes"><xref rid="bi020397eb00024" ref-type="bibr"></xref></italic>). GST pull-down assays using glutathione−Sepharose-bound GST or GST-P58<sup>IPK</sup>, and 1 × 10<sup>5</sup> cpm of radiolabeled
P52<sup>rIPK</sup> translation products, were conducted and analyzed
exactly as described (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">Yeast Methods. </italic>The yeast two-hybrid assay was used to
identify in vivo protein−protein interactions as described
previously (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00024" ref-type="bibr"></xref>, <xref rid="bi020397eb00030" ref-type="bibr"></xref></named-content></italic>). Plasmids encoding GAL4 AD or BD
fusion proteins were introduced into <italic toggle="yes">S. cerevisiae </italic>Hf7c
[<italic toggle="yes">MATa ura3-52 hi3-200 lys2-801 ade2-101 trp1-901 leu2-3, 112 gal4-542 gal80-538 LYS2::GAL1-HIS3 URA3(GAL4
17-mers)<sub>3</sub></italic><italic toggle="yes">-CYC-lacZ</italic>; Clontech]. Transformants were plated
onto synthetic defined (SD) medium supplemented with
histidine (His<sup>+</sup>), but lacking leucine and tryptophan, to select
for the retention of the pAD and pBD plasmids. Colonies
were subsequently streaked onto SD medium lacking His,
Trp, and Leu (His<sup>-</sup>) and grown at 30 °C for 3 days to deplete
endogenous histidine stores. Colonies were then replica
printed onto His<sup>+</sup> and His<sup>-</sup> medium and incubated at 30 °C
for 2−4 days. Colonies were scored positive for a two-hybrid
protein interaction if they exhibited growth on both His<sup>-</sup> and
His<sup>+</sup> medium. Liquid β-Gal assays, using the fluorogenic
substrate 4-methylumbelliferyl β-<sc>d</sc>-galactoside (MUG; Sigma),
were conducted on yeast two-hybrid strains as previously
reported (<italic toggle="yes"><xref rid="bi020397eb00024" ref-type="bibr"></xref></italic>).
</p><p>To assess protein function in vivo, pYex4-P58<sup>IPK</sup> and pYX-P52<sup>rIPK</sup> wild-type and mutant constructs were introduced into
<italic toggle="yes">S. cerevisiae </italic>RY1-1 [<italic toggle="yes">MATa ura3-52 leu2-3 leu2-112 gcn2Δ
trp1-Δ63 LEU2::(GAL-CYC1-PKR</italic>)<sub>2</sub>] (<italic toggle="yes"><xref rid="bi020397eb00034" ref-type="bibr"></xref></italic>). This strain lacks
the endogneous yeast eIF2α kinase, GCN2, but contains two
copies of human <italic toggle="yes">PKR</italic> integrated into the <italic toggle="yes">LEU2 </italic>locus under
control of the galactose-inducible <italic toggle="yes">GAL</italic>/<italic toggle="yes">CYC1</italic> hybrid promoter. In the presence of galactose, PKR is expressed and
phosphorylates eIF2α, resulting in suppression of cell growth.
However, the coexpression of PKR regulatory proteins results
in modulation of PKR function, which can be scored by the
amount of galactose-specific growth and by analysis of eIF2α
phosphorylation (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00030" ref-type="bibr"></xref>, <xref rid="bi020397eb00034" ref-type="bibr"></xref></named-content></italic>). RY1-1 transformants were plated
onto SD medium lacking uracil and tryptophan and incubated
at 30 °C for 2−4 days. Resultant colonies were plated onto
synthetic defined galactose (SGal) medium (containing 2%
galactose as the sole carbon source), incubated for 5−9 days
at 30 °C, and scored for growth as described previously (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">Isoelectric Focusing Analysis.</italic> Isoelectric focusing (IEF)
analysis to determine the phosphorylation state of yeast
eIF2α was carried out essentially as described (<italic toggle="yes"><xref rid="bi020397eb00035" ref-type="bibr"></xref></italic>). Approximately 20 μg of total protein from each extract was
separated by single-dimension IEF and blotted to nitrocellulose membranes. Blots were probed with rabbit polyclonal
antiserum specific to yeast eIF2α.
</p></sec><sec id="d7e884"><title>Results</title><p><italic toggle="yes">The Charged Domain of P52<sup>rIPK</sup></italic><sup></sup><italic toggle="yes"> Is Necessary and Sufficient for Interaction with P58<sup>IPK</sup></italic><sup></sup><italic toggle="yes">. </italic>The charged domain of
P52<sup>rIPK</sup> exhibits homology to the charged domains of Hsp90,
which include an N-terminal geldanamycin-binding domain
and a region located at the C terminus of the protein (<italic toggle="yes">30</italic>,<italic toggle="yes">
36</italic>,<italic toggle="yes"> 37</italic>). Both Hsp90 and P52<sup>rIPK</sup> bind to TPR proteins, and
in the case of Hsp90, the charged domains appear to be TPR
domain acceptor sites that mediate protein−protein interactions (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00038" ref-type="bibr"></xref>, <xref rid="bi020397eb00039" ref-type="bibr"></xref></named-content></italic>). In previous work, we demonstrated that P52<sup>rIPK</sup>
binds P58<sup>IPK</sup> and inhibits the PKR regulatory properties of
P58<sup>IPK</sup> in vitro and in vivo (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>). To understand the mechanism by which P52<sup>rIPK</sup> regulates P58<sup>IPK</sup> and PKR function,
we conducted detailed structure−function analyses of the
P52<sup>rIPK</sup>−P58<sup>IPK</sup> interaction. We hypothesized that the P52<sup>rIPK</sup>
charged domain might directly bind to P58<sup>IPK</sup>, possibly in a
TPR domain-dependent fashion.
</p><p>In initial experiments, we measured the relative binding
of in vitro translated wild-type P52<sup>rIPK</sup>, and progressive
C-terminal P52<sup>rIPK</sup> truncation products, to GST-P58<sup>IPK</sup> in a
cell-free GST pull-down assay. As shown in Figure <xref rid="bi020397ef00001"></xref>A, full-length P52<sup>rIPK</sup>, and P52<sup>rIPK</sup> aa 1−384 in vitro translation
products, bound efficiently in a dose-dependent manner to
immobilized GST-P58<sup>IPK</sup> but not to immobilized GST alone.
P52<sup>rIPK</sup> aa 1−239 translation products retained the ability to
specifically bind to GST-P58<sup>IPK</sup>, albeit at a reduced level
(Figure <xref rid="bi020397ef00001"></xref>A, lanes 9−12). These results indicate that the
P58<sup>IPK</sup>-binding activity resides within the N-terminal region
of P52<sup>rIPK</sup> and that aa 1−239, which include the charged
domain, are sufficient to mediate interaction with P58<sup>IPK</sup> in
vitro. Variations in the ability of the different P52<sup>rIPK</sup> deletion
constructs to bind to P58<sup>IPK</sup> could be due to conformation
changes resulting from protein truncation.
<fig id="bi020397ef00001" position="float" orientation="portrait"><label>1</label><caption><p>Mapping the P58<sup>IPK</sup> interactive domain of P52<sup>rIPK</sup>. (A) GST pull-down assay. Equal molar amounts of GST or GST-P58<sup>IPK</sup> were
immobilized on glutathione−Sepharose beads, and increasing amounts of bead-bound complexes were mixed with 1 × 10<sup>5</sup> cpm of <sup>35</sup>S-labeled in vitro translation products corresponding to full-length P52<sup>rIPK</sup> (aa 1−492; lanes 1−4), P52<sup>rIPK</sup> aa 1−384 (lanes 5−8), or P52<sup>rIPK</sup>
aa 1−239 (lanes 9−12). After the beads were washed, protein complexes were eluted and then separated by SDS−PAGE. An autoradiogram
of the dried gel is shown. The arrow points to the 1−239 translation product. The positions of protein standards are indicated in kDa on
the left. The lower panel shows the amount of input P52<sup>rIPk</sup> translation products used in the binding assays. (B) Schematic representation
of wild-type P52<sup>rIPK</sup> (top) and P52<sup>rIPK</sup> deletion mutants used in mapping the P58<sup>IPK</sup> interactive domain by yeast two-hybrid analysis. Each
construct was expressed in<italic toggle="yes"> S. cerevisiae</italic> Hf7c as a GAL4 activation domain fusion and is identified by the encoded aa residues shown on
the left. The dark region denotes the 114 aa charged domain of P52<sup>rIPK</sup> that exhibits homology to Hsp90 (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>). This region is deleted from
the Δ85−200 construct. The results from P58<sup>IPK</sup> interaction studies are summarized on the right. (C) Expression of AD-P52<sup>rIPK</sup> fusion
proteins in yeast. Extracts from yeast coexpressing BD-P58<sup>IPK</sup> and the indicated AD constructs were subjected to immunoblot analysis
using an antibody specific to the GAL4 activation domain. Lane 1 shows an extract from yeast coexpressing the AD vector. Lanes 2−8
show extracts from yeast coexpressing AD fusion proteins corresponding to P52<sup>rIPK</sup> aa 1−492 (wild type, lane 2), 237−492 (lane 3),
1−243 (lane 4), 1−84 (lane 5), 86−203 (lane 6), 1−203 (lane 7), and Δ85−200 (lane 8). The positions of protein standards are shown on
the left. (D) Yeast two-hybrid assay. Hf7c yeast cells coexpressing the indicated AD fusion proteins with the GAL4 DNA-binding domain
alone (BD vector), or BD-P58<sup>IPK</sup>, were replica printed onto medium in the presence (+HIS, left panel) or absence (−HIS, right panel) of
histidine and incubated at 30 °C for 4 days. In this assay, growth on both His<sup>+</sup> and His<sup>-</sup> medium is indicative of a two-hybrid protein
interaction (<italic toggle="yes"><xref rid="bi020397eb00024" ref-type="bibr"></xref></italic>). To confirm the growth phenotype, we conducted liquid β-Gal assays of the encoded β-Gal reporter using the MUG substrate.
Results are shown to the right of each panel and are the average of three separate experiments. Numbers shown were rounded to the nearest
whole value.</p></caption><graphic xlink:href="bi020397ef00001.tif" position="float" orientation="portrait"></graphic></fig></p><p>To verify the in vitro binding data, and to test the role of
the 114 aa charged domain in mediating interaction with
P58<sup>IPK</sup>, we constructed a series of P52<sup>rIPK</sup> deletion mutants
for use in a yeast two-hybrid in vivo interaction assay (Figure
<xref rid="bi020397ef00001"></xref>B). Each construct was coexpressed as a GAL4 activation
domain (AD) fusion protein in <italic toggle="yes">S. cerevisiae</italic> strains expressing the GAL4 DNA-binding domain (BD) or a BD-P58<sup>IPK</sup>
fusion protein. Each AD-P52<sup>rIPK</sup> construct was efficiently
expressed (Figure <xref rid="bi020397ef00001"></xref>C) and was scored for its ability to
specifically bind to BD-P58<sup>IPK</sup> by its ability to promote the
growth of each yeast strain on His<sup>-</sup> medium. Whereas all
stains grew equally well on His<sup>+</sup> medium (Figure <xref rid="bi020397ef00001"></xref>D, left
panel), exclusion of the P52<sup>rIPK</sup> charged domain resulted in
abrogation of P58<sup>IPK</sup> interaction and a lack of growth on His<sup>-</sup>
medium (Figure <xref rid="bi020397ef00001"></xref>D, right panel). In particular, P52<sup>rIPK</sup> aa
86−203 were sufficient to mediate interaction with P58<sup>IPK</sup>,
since yeast cells expressing this construct maintained robust
growth on His<sup>-</sup> medium. Conversely, the P52<sup>rIPK</sup> Δ85−200
construct, which lacks the charged domain, failed to confer
growth on selective medium, even though this construct was
efficiently expressed (see Figure <xref rid="bi020397ef00001"></xref>C,D). Analysis of the
encoded β-Gal activity (MUG units in Figure <xref rid="bi020397ef00001"></xref>D) within
each strain confirmed the growth assay data and indicated
that P52<sup>rIPK</sup> aa 86−203 bound to P58<sup>IPK</sup> at a relative strength
approximating or slightly surpassing that of the wild-type
protein. Taken together, these studies demonstrate that the
charged domain of P52<sup>rIPK</sup> is both necessary and sufficient
for interaction with P58<sup>IPK</sup>. The localization of P58<sup>IPK</sup>-binding
activity to within the charged domain of P52<sup>rIPK</sup> is of interest,
since the charged acidic domains in the N- and C-terminal
regions of Hsp90 also interact with TPR proteins (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00038" ref-type="bibr"></xref>−<xref rid="bi020397eb00039" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi020397eb00040" ref-type="bibr"></xref></named-content></italic>).
</p><p><italic toggle="yes">P58<sup>IPK</sup></italic><sup></sup><italic toggle="yes"> TPR Domain 7 Is Necessary and Sufficient for
Interaction with P52<sup>rIPK</sup></italic><sup></sup><italic toggle="yes">.</italic> TPR domains are modular protein
interaction motifs that are present in a diverse family of
proteins that includes many cell-signaling effectors (<italic toggle="yes"><xref rid="bi020397eb00041" ref-type="bibr"></xref></italic>). As
shown in Figure <xref rid="bi020397ef00002"></xref>, P58<sup>IPK</sup> contains nine tandemly arranged
TPR motifs (<italic toggle="yes"><xref rid="bi020397eb00021" ref-type="bibr"></xref></italic>). We previously demonstrated that TPR
domain 6 directs the interaction between P58<sup>IPK</sup> and PKR
(<italic toggle="yes"><xref rid="bi020397eb00024" ref-type="bibr"></xref></italic>). To determine if the interaction with P52<sup>rIPK</sup> was
similarly TPR domain dependent, we conducted a series of
protein interaction studies using the yeast two-hybrid system
to score for protein interaction in vivo. GAL4 DNA-binding
domain fusion proteins of wild-type P58<sup>IPK</sup>, or various P58<sup>IPK</sup>
deletion constructs, were coexpressed with the GAL4 activation domain or the AD-P52<sup>rIPK</sup> wild-type fusion protein, and
each strain was scored for growth on His<sup>-</sup> medium. The
relative strength of the two-hybrid protein interactions was
scored by β-Gal assay (MUG units). In initial experiments,
we tested a series of P58<sup>IPK</sup> N- and C-terminal deletion
constructs for their ability to bind P52<sup>rIPK</sup>. Expression of BD
fusion proteins P58<sup>IPK</sup> 8−4 or 9−1, which respectively
encode P58<sup>IPK</sup> aa 1−302 or 168−504, was sufficient for
interaction with AD-P52<sup>rIPK</sup>, as determined by growth of the
corresponding strains on His<sup>-</sup> medium (Figure <xref rid="bi020397ef00002"></xref>). In contrast,
P58<sup>IPK</sup> aa 1−166, encoded by the BD-P58<sup>IPK</sup> 8−1 protein,
failed to mediate interaction with AD-P52<sup>rIPK</sup>. From these
initial studies, we preliminarily mapped the P52<sup>rIPK</sup> interactive
domain of P58<sup>IPK</sup> to within aa 168−302, which extends from
midway within TPR domain 4 through TPR domain 7 (Figure
<xref rid="bi020397ef00002"></xref>A).
<fig id="bi020397ef00002" position="float" orientation="portrait"><label>2</label><caption><p>Mapping the P52<sup>rIPK</sup> interacting domain of P58<sup>IPK</sup>. (A)
Structural representation of P58<sup>IPK</sup> and TPR domain deletion
constructs. TPR domains are indicated by number in gray-shaded
squares, and aa positions defining TPR domain 7 are indicated.
Amino acid positions are shown to denote full-length P58<sup>IPK</sup>, the
terminal aa positions within the P58<sup>IPK</sup> 8−1, 8−4, 9−1, 10−1, and
TPR7 truncation mutants, and the positions of deleted TPR domain
5 (ΔTPR5), 6 (ΔTPR6), and 7 (ΔTPR7). The results from protein
interaction studies with P52<sup>rIPK</sup> are summarized on the right, together
with β-Gal assay results (in MUG units). Values shown are the
average of two independent experiments and are rounded to the
nearest whole number. (B) Analysis of BD-P58<sup>IPK</sup> and TPR deletion
mutants in the yeast two-hybrid assay. Hf7c yeast cells coexpressing
the indicated BD-P58<sup>IPK</sup> fusion proteins and AD-P52<sup>rIPK</sup> or the
GAL4 activation domain alone (AD vector) were streaked onto His<sup>-</sup>
medium, and plates were incubated for 4 days at 30 °C. Growth is
indicative of a two-hybrid protein interaction. All AD and BD fusion
proteins were expressed in each strain, and all strains grew
efficiently on control His<sup>+</sup> medium (data not shown).</p></caption><graphic xlink:href="bi020397ef00002.tif" position="float" orientation="portrait"></graphic></fig></p><p>To further define the P52<sup>rIPK</sup> interactive domain of P58<sup>IPK</sup>,
we tested the ability of P58<sup>IPK</sup> TPR domain deletion mutants,
lacking either TPR domains 5, 6, or 7 (encoded in the BD-P58<sup>IPK</sup> ΔTPR 5, ΔTPR6, and ΔTPR 7 fusion constructs,
respectively), to interact with P52<sup>rIPK</sup>. Each construct was
coexpressed in yeast with either the GAL4 activation domain
or AD-P52<sup>rIPK</sup>. Deletion of TPR domain 6 did not affect
P58<sup>IPK</sup> binding to P52<sup>rIPK</sup>. Indeed, yeast cells coexpressing
AD-P52<sup>rIPK</sup> with wild-type P58<sup>IPK</sup>, ΔTPR 5, or ΔTPR 6 BD
fusion proteins exhibited robust growth on His<sup>-</sup> medium,
and the encoded β-Gal activity approximated that of the wild-type P58<sup>IPK</sup> protein. In contrast, the 10−1 construct (encoding
TPR domains 4−6 and the flanking 12 aa) failed to interact
with P52<sup>rIPK</sup>. Similarly, deletion of TPR domain 7 ablated
the ability of P58<sup>IPK</sup> to bind P52<sup>rIPK</sup> in this assay (Figure <xref rid="bi020397ef00002"></xref>).
The 34 -aa TPR domain 7 was sufficient to mediate an
interaction with P52<sup>rIPK</sup> that was equivalent in strength (MUG
units) to the wild-type protein. We confirmed that all BD
and AD constructs were efficiently expressed in the corresponding yeast cells (data not shown). The ΔTPR 5 and
ΔTPR 7 constructs retained their ability to bind PKR in
parallel yeast two-hybrid studies (data not shown), indicating
that deletion of each TPR domain had little impact on overall
protein conformation. These results demonstrate that P58<sup>IPK</sup>
TPR domain 7 is both necessary and sufficient for interaction
with P52<sup>rIPK</sup> in vivo.
</p><p><italic toggle="yes">P58<sup>IPK</sup></italic><sup></sup><italic toggle="yes"> TPR Domain 7 Is Required for P52<sup>rIPK</sup></italic><sup></sup><italic toggle="yes">-Dependent
Regulation of PKR. </italic>Our results demonstrate that P58<sup>IPK</sup> binds
to PKR and P52<sup>rIPK</sup> at independent, but adjacent, sites within
TPR domains 6 and 7, respectively. We previously showed
that P58<sup>IPK</sup> inhibition of PKR requires TPR domain 6 (<italic toggle="yes"><xref rid="bi020397eb00024" ref-type="bibr"></xref></italic>)
and that this likely occurs through formation of a TPR
6-mediated P58<sup>IPK</sup>-PKR inhibitory complex in vivo (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00022" ref-type="bibr"></xref>, <xref rid="bi020397eb00025" ref-type="bibr"></xref></named-content></italic>).
We sought to determine if P52<sup>rIPK</sup> control of P58<sup>IPK</sup> was
similarly a TPR-dependent event, possibly mediated by the
P52<sup>rIPK</sup> interaction with P58<sup>IPK</sup> TPR domain 7. For these
experiments, we utilized a yeast genetic system to assess
the role of P52<sup>rIPK</sup> in the P58<sup>IPK</sup>-dependent regulation of PKR
and eIF2α phosphorylation (<italic toggle="yes"><xref rid="bi020397eb00034" ref-type="bibr"></xref></italic>). This experimental system
is based upon the observation that PKR is growth suppressive
when expressed in yeast, due to high levels of PKR-mediated
eIF2α phosphorylation (<italic toggle="yes"><xref rid="bi020397eb00035" ref-type="bibr"></xref></italic>). We employed the gcn2Δ yeast
strain RY1-1, which lacks the endogenous yeast eIF2α kinase
but harbors two integrated copies of human PKR placed
under the transcriptional control of a galactose-inducible
promoter (<italic toggle="yes"><xref rid="bi020397eb00034" ref-type="bibr"></xref></italic>). This strain grows well when streaked onto
noninducing dextrose medium but exhibits a slow growth
phenotype when streaked onto galactose medium, due to
induced expression and activity of PKR.
</p><p>We previously demonstrated that, when expressed in RY1-1, P58<sup>IPK</sup> represses PKR-mediated eIF2α phosphorylation
and relieves PKR-mediated growth suppression (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00022" ref-type="bibr"></xref>, <xref rid="bi020397eb00030" ref-type="bibr"></xref></named-content></italic>).
However, the introduction of P52<sup>rIPK</sup> into RY1-1 expressing
both PKR and P58<sup>IPK</sup> results in restoration of PKR activity
and eIF2α phosphorylation, concomitant with growth suppression due to inhibition of P58<sup>IPK</sup> function (<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>). The effects
of this reconstituted PKR regulatory pathway on yeast growth
are shown in Figure <xref rid="bi020397ef00003"></xref>A. RY1-1 harboring vectors alone (V/V), or in combination with P52<sup>rIPK</sup> (V/P52<sup>rIPK</sup>), failed to grow
when streaked onto galactose medium. Expression of P58<sup>IPK</sup>
was sufficient to reverse PKR-mediated growth suppression
(Figure <xref rid="bi020397ef00003"></xref>A, P58<sup>IPK</sup>/V), confirming that P58<sup>IPK</sup> can repress
PKR function in yeast (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00022" ref-type="bibr"></xref>, <xref rid="bi020397eb00030" ref-type="bibr"></xref></named-content></italic>). In comparison, the introduction of P52<sup>rIPK</sup> into this strain restored the slow growth
phenotype (Figure <xref rid="bi020397ef00003"></xref>A, compare P58<sup>IPK</sup>/V and P58<sup>IPK</sup>/P52<sup>rIPK</sup>
strains), again demonstrating that P52<sup>rIPK</sup> can inhibit P58<sup>IPK</sup>
function to restore PKR activity in vivo. RY1-1 expressing
P58<sup>IPK</sup> that lacked TPR domain 7 (P58<sup>IPK</sup>Δ7/V) exhibited
growth on galactose medium, indicating that deletion of TPR
domain 7 does not hinder the PKR regulatory properties of
P58<sup>IPK</sup>. P52<sup>rIPK</sup> coexpression had no effect on the growth of
this strain on galactose medium (compare P58<sup>IPK</sup> Δ7/V with
P58<sup>IPK</sup> Δ7/P52<sup>rIPK</sup>, Figure <xref rid="bi020397ef00003"></xref>A). Examination of protein
expression in these cells revealed that each expression
construct, and PKR, was efficiently expressed when the cells
were grown on galactose medium (Figure <xref rid="bi020397ef00003"></xref>B). It is important
to note that P52<sup>rIPK</sup> itself is not toxic when expressed in yeast
(<italic toggle="yes"><xref rid="bi020397eb00030" ref-type="bibr"></xref></italic>), and this observation was confirmed using RY1-1
coexpressing P58<sup>IPK</sup> Δ7 with P52<sup>rIPK</sup>. Our results therefore
demonstrate that TPR domain 7 of P58<sup>IPK</sup> is required for
control of P58<sup>IPK</sup> function by P52<sup>rIPK</sup>. When taken together
with our protein interaction data, these studies indicate that
P52<sup>rIPK</sup> mediates control of P58<sup>IPK</sup>, and thereby regulation
of PKR, by binding to P58<sup>IPK</sup> TPR domain 7. In this manner,
P52<sup>rIPK</sup> releases the block to PKR activity imposed by P58<sup>IPK</sup>,
resulting in inhibition of growth on galactose medium (Figure
<xref rid="bi020397ef00003"></xref>A).
<fig id="bi020397ef00003" position="float" orientation="portrait"><label>3</label><caption><p>P52<sup>rIPK</sup> mediates control of P58<sup>IPK</sup> and PKR through P58<sup>IPK</sup> TPR domain 7. (A) Yeast growth assay for the analysis of PKR
regulation in vivo. RY1-1 yeast cells were streaked onto selective SGal medium to induce coexpression of PKR and the indicated P58<sup>IPK</sup>
and P52<sup>rIPK</sup> alleles. The plate shown was incubated for 6 days at 30 °C. In this assay, yeast growth is indicative of PKR inhibition. Growth
suppression indicates that PKR is active. All strains exhibited efficient growth on SD medium (not shown). V denotes the empty expression
vector in place of P58<sup>IPK</sup> or P52<sup>rIPK</sup>. (B) Protein expression in RY1-1 yeast cells. Extracts from the yeast cells shown in panel A were
subjected to immunoblot analysis with antibodies specific to human PKR (top panel), P58<sup>IPK</sup> (second panel from top), P52<sup>rIPK</sup> (third panel
from top), or actin (lower panel). Results were reproducible over three experiments and are shown from a single blot that was first probed
with antiserum to P52<sup>rIPK</sup>, stripped, and then sequentially reprobed with each individual antibody. Lanes show protein expression in extracts
prepared from yeast cells coexpressing PKR with P58<sup>IPK</sup> Δ7 (lane 1), P58<sup>IPK</sup> Δ7 and P52<sup>rIPK</sup> (lane 2), vectors only (V/V, lane 3), P52<sup>rIPK</sup>
(lane 4), P58<sup>IPK</sup> (lane 5), or P58<sup>IPK</sup> and P52<sup>rIPK</sup> (lane 6). Arrowheads at right indicate the positions of wild-type P58<sup>IPK</sup>, P58<sup>IPK</sup> Δ7, and
P52<sup>rIPK</sup>.
</p></caption><graphic xlink:href="bi020397ef00003.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">The Charged Domain of P52<sup>rIPK</sup></italic><sup></sup><italic toggle="yes"> Is Necessary and Sufficient for Control of P58<sup>IPK</sup></italic><sup></sup><italic toggle="yes"> Function in Vivo.</italic> Our protein
interaction data demonstrated that the charged domain of
P52<sup>rIPK</sup>, encoding aa 86−200, was both necessary and
sufficient to mediate interaction with P58<sup>IPK</sup> in vivo (see
Figure <xref rid="bi020397ef00001"></xref>). We therefore sought to determine if this region
of P52<sup>rIPK</sup> was similarly required for protein function. When
coexpressed with P58<sup>IPK</sup> in RY1-1, a P52<sup>rIPK</sup> mutant lacking
the entire 114 aa charged domain (P52<sup>rIPK</sup> Δ85−200) failed
to inhibit P58<sup>IPK</sup> function, as demonstrated by growth on
galactose medium (Figure <xref rid="bi020397ef00004"></xref>, P58<sup>IPK</sup>/P52<sup>rIPK</sup> Δ85−200). This
is in stark contrast to the ability of wild-type P52<sup>rIPK</sup> to inhibit
P58<sup>IPK</sup> function and restore the PKR growth-suppressive
phenotype (Figure <xref rid="bi020397ef00004"></xref>, compare P58<sup>IPK</sup>/P52<sup>rIPK</sup> to P58<sup>IPK</sup>/P52<sup>rIPK</sup> Δ85−200). As a control for potential construct
toxicity, we confirmed that P52<sup>rIPK</sup> Δ85−200 had no effect
on P58<sup>IPK</sup> Δ7-dependent growth rescue (P58<sup>IPK</sup> Δ7/P52<sup>rIPK</sup>
Δ85−200). Similarly, RY1-1 expressing either P58<sup>IPK</sup> or
P58<sup>IPK</sup> Δ7 exhibited growth on galactose, indicative of P58<sup>IPK</sup>
repression of PKR (Figure <xref rid="bi020397ef00004"></xref>). Coexpression of P52<sup>rIPK</sup> aa
86−200 was sufficient to block the PKR inhibitory function
of wild-type P58<sup>IPK</sup> but not of the P58<sup>IPK</sup> ΔTPR7 mutant
(Figure <xref rid="bi020397ef00004"></xref>, lower panel). We again confirmed that all
constructs were efficiently expressed (data not shown). Thus,
P52<sup>rIPK</sup> aa 86−200 are both necessary and sufficient to
suppress the PKR regulatory function of P58<sup>IPK</sup>, demonstrating that the charged domain of P52<sup>rIPK</sup> is required for
inhibition of P58<sup>IPK</sup> in vivo.
<fig id="bi020397ef00004" position="float" orientation="portrait"><label>4</label><caption><p>The charged domain of P52<sup>rIPK</sup> is necessary and
sufficient for the control of P58<sup>IPK</sup> and regulation of PKR function
in vivo. RY1-1 yeast cells were streaked onto selective SGal
medium to induce coexpression of PKR with the indicated P58<sup>IPK</sup>
and P52<sup>rIPK</sup> alleles. The panels shown are of plates that were
incubated at 30 °C for 7 days. V denotes the empty expression
vector in place of P58<sup>IPK</sup> or P52<sup>rIPK</sup>. Growth is indicative of PKR
inhibition by P58<sup>IPK</sup>. All strains exhibited efficient growth on SD
medium (data not shown).</p></caption><graphic xlink:href="bi020397ef00004.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">P58<sup>IPK</sup></italic><sup></sup><italic toggle="yes"> TPR Domain 7 and the Charged Domain of P52<sup>rIPK</sup></italic><sup></sup><italic toggle="yes">
Are Required To Control eIF2α Phosphorylation by PKR.</italic>
As upstream regulators of PKR function, P52<sup>rIPK</sup> and P58<sup>IPK</sup>
are proposed to modulate the phosphorylation of PKR
substrates, including eIF2α. To determine how the P58<sup>IPK</sup>−P52<sup>rIPK</sup> interaction may influence eIF2α phosphorylation, we
examined the levels of endogenous eIF2α in yeast cells
coexpressing PKR and various forms of P58<sup>IPK</sup> and P52<sup>rIPK</sup>.
We used a sensitive IEF procedure to separate the PKR
phosphorylated isoform of eIF2α (phosphorylated on serine
51) from the non-PKR phosphorylated eIF2α isoform in
strains grown in liquid SGal media. Induction of PKR
expression resulted in high levels of eIF2α phosphorylation
(Figure <xref rid="bi020397ef00005"></xref>, compare lanes 1 and 2) and was not significantly
affected by expression of P52<sup>rIPK</sup> (lane 8) or P52<sup>rIPK</sup> Δ85−200 (lane 3). In contrast, expression of P58<sup>IPK</sup> resulted in an
accumulation of the hypophosphorylated eIF2α species.
Although the majority of eIF2α remained phosphorylated
in the presence of P58<sup>IPK</sup>, we have observed that relatively
small changes in the overall level of eIF2α phosphorylation
can have a significant effect on cell growth (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00030" ref-type="bibr"></xref>, <xref rid="bi020397eb00042" ref-type="bibr"></xref></named-content></italic>). The
coexpression of wild-type P52<sup>rIPK</sup> blocked the P58<sup>IPK</sup>-mediated reduction of eIF2α phosphorylation (Figure <xref rid="bi020397ef00005"></xref>,
compare lanes 4 and 5), whereas coexpression of P52<sup>rIPK</sup>
lacking the charged domain had no effect on P58<sup>IPK</sup> function
(lane 7). This result confirms that the 114 aa charged domain
is responsible for the downstream regulation of P58<sup>IPK</sup> and
PKR. When compared to cells expressing wild-type P58<sup>IPK</sup>,
cells expressing P58<sup>IPK</sup> Δ7 exhibited a marked increase in
the accumulation of hypophosphorylated eIF2α. Moreover,
this reduction in eIF2α phosphorylation was maintained even
in the presence of P52<sup>rIPK</sup> (Figure <xref rid="bi020397ef00005"></xref>, lane 6). In general, we
found that cells expressing P58<sup>IPK</sup> Δ7 exhibited significantly
shorter doubling times when grown in the presence of
galactose (data not shown). These results suggest that TPR
domain 7 confers negative regulation to P58<sup>IPK</sup> and that
deletion of this domain results in hyperactive protein
function. When taken together with the protein interaction
and cell growth data, our analysis of eIF2α phosphorylation
demonstrates that the 114 aa charged domain of P52<sup>rIPK</sup>
regulates P58<sup>IPK</sup> through an interaction with P58<sup>IPK</sup> TPR
domain 7 and that this interaction modulates PKR-mediated
eIF2α phosphorylation.
<fig id="bi020397ef00005" position="float" orientation="portrait"><label>5</label><caption><p>eIF2α phosphorylation analysis. Yeast extracts were separated by single-dimension IEF and blotted to nitrocellulose, and eIF2α was detected by immunoblot analysis. This procedure allows for the discrimination and detection of the serine 51 phosphorylated eIF2α isoform (phosphorylated by PKR) from the less acidic, basely phosphorylated eIF2α isoform (<italic toggle="yes"><xref rid="bi020397eb00035" ref-type="bibr"></xref></italic>). Lanes 1 and
2 correspond to extracts from RY1-1 control cells harboring the
expression plasmids alone and grown in SGal or SD medium to
induce (+PKR) or suppress PKR expression (−PKR). Lanes 3−8
correspond to extracts prepared from RY1-1 cells grown in SGal
medium and coexpressing PKR with P52<sup>rIPK</sup> Δ85−200 (lane 3),
P58<sup>IPK</sup> (lane 4), P58<sup>IPK</sup> and P52<sup>rIPK</sup> (lane 5), P58<sup>IPK</sup> Δ7 and P52<sup>rIPK</sup>
(lane 6), P58<sup>IPK</sup> and P52<sup>rIPK</sup> Δ85−200 (lane 7), or P52<sup>rIPK</sup> (lane 8).
Arrows at the right denote the positions of basely phosphorylated
eIF2α and the serine 51 phosphorylated eIF2α isoforms.</p></caption><graphic xlink:href="bi020397ef00005.tif" position="float" orientation="portrait"></graphic></fig></p><p><italic toggle="yes">Regulation of the P52<sup>rIPK</sup></italic><sup></sup><italic toggle="yes">−P58<sup>IPK</sup></italic><sup></sup><italic toggle="yes"> Complex in HeLa Cells.</italic>
Our results indicate that P52<sup>rIPK</sup> and P58<sup>IPK</sup> mediate a
regulatory pathway that responds to stress-induced signals.
This notion is supported by our previous work that defined
P58<sup>IPK</sup> as a stress-activated cochaperone and regulator of
PKR (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00023" ref-type="bibr"></xref>, <xref rid="bi020397eb00027" ref-type="bibr"></xref></named-content></italic>). We therefore sought to determine if the
formation of a P52<sup>rIPK</sup>−P58<sup>IPK</sup> complex was regulated in
response to cellular stress. We began by analyzing this
interaction in HeLa cells that were deprived of serum for a
period of 4 h in order to induce an acute stress response.
Cell extracts prepared from serum-starved and control
cultures were then subjected to immunoprecipitation analysis
to assess endogenous complex formation between P58<sup>IPK</sup> and
P52<sup>rIPK</sup>. To detect complex formation, anti-P52<sup>rIPK</sup> immunoprecipitation products were recovered and subjected to
immunoblot analysis with a Mab specific to P58<sup>IPK</sup>. As
shown in Figure <xref rid="bi020397ef00006"></xref>A, we failed to detect significant complex
formation between P58<sup>IPK</sup> and P52<sup>rIPK</sup> within control HeLa
cells cultured in complete medium. However, removal of
serum from the growth media induced the association of
P58<sup>IPK</sup> with P52<sup>rIPK</sup>, and P58<sup>IPK</sup> was recovered within anti-P52<sup>rIPK</sup> immunoprecipitation reactions (Figure <xref rid="bi020397ef00006"></xref>A, compare
lanes 2 and 3). No detectable changes in the level of P52<sup>rIPK</sup>
protein were observed between control and serum-starved
cultures (data not shown). Importantly, P58<sup>IPK</sup> was not
present in control immunoprecipitation products recovered
from serum-starved cells using preimmune rabbit control
serum (Figure <xref rid="bi020397ef00006"></xref>A, lane 1). These results demonstrate that
P58<sup>IPK</sup> and P52<sup>rIPK</sup> form a stable complex in cells stressed
by serum deprivation.
<fig id="bi020397ef00006" position="float" orientation="portrait"><label>6</label><caption><p>Stress-regulated complex formation of P52<sup>rIPK</sup> and P58<sup>IPK</sup>
in HeLa cells. (A) HeLa cells were cultured for 4 h in the presence
(lanes 2 and 5) or absence of serum (lanes 1, 3, and 4). Extracts
were prepared, and 600 μg of protein was subjected to immunoprecipitation with normal (preimmune) rabbit serum (nrs, lane 1)
or anti-P52<sup>rIPK</sup> rabbit serum (lanes 2 and 3). After washing, the
immunoprecipitation products were resolved by electrophoresis in
a 10% acrylamide gel and subjected to immunoblot analysis with
a P58<sup>IPK</sup>-specific Mab. To adequately separate the immunoglobulin
heavy chain (dark smear) from the P58<sup>IPK</sup> band, the electrophoresis
was conducted such that the 55 kDa standard was resolved to very
near the bottom of the gel. The positions of the 68 and 55 kDa
protein standards are shown on the left. Lanes 4 and 5 show P58<sup>IPK</sup>
and P52<sup>rIPK</sup> (arrowheads, upper and lower panels, respectively) in
50 μg of the input extract that was used in the immunoprecipitation
reactions. (B) Cells were untreated (lane 1), serum starved for 4 h
(lane 2), heat shocked (lane 3), or cultured with sodium arsenite
for 2 h (lane 4), thapsigargin for 4 h (lane 5), or tunicamycin for
4 h (lane 6), and harvested, and 400 μg of each extract was subjected
to immunoprecipitation with α-P52<sup>rIPK</sup> serum as described above.
P58<sup>IPK</sup> was identified in the immunocomplexes by Western blot
analysis (upper panel), and arrows point to the positions of the
recovered P58<sup>IPK</sup> or immunoglobulin (Ig) heavy chain. The lower
panels show P52<sup>rIPK</sup> and P58<sup>IPK</sup> protein levels in 50 μg of the input
extract from each immunoprecipitation reaction.</p></caption><graphic xlink:href="bi020397ef00006.tif" position="float" orientation="portrait"></graphic></fig></p><p>We next examined P52<sup>rIPK</sup>−P58<sup>IPK</sup> complex formation in
response to heat shock, or treatment with sodium arsenite,
thapsigargin, or tunicamycin. As shown in Figure <xref rid="bi020397ef00006"></xref>B, serum
withdrawal, heat shock, and treatment with sodium arsenite
all resulted in the induction of a stable P52<sup>rIPK</sup>−P58<sup>IPK</sup>
complex. In contrast, we were unable to detect significant
levels of the P52<sup>rIPK</sup>−P58<sup>IPK</sup> complex in cells treated with
thapsigargin or tunicamycin. Serum withdrawal, heat shock,
and sodium arsenite treatment each trigger cytoplasmic stress-response programs, whereas thapsigargin and tunicamycin
are specific inducers of ER stress (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00001" ref-type="bibr"></xref>, <xref rid="bi020397eb00043" ref-type="bibr"></xref></named-content></italic>). The induction of
the P52<sup>rIPK</sup>−P58<sup>IPK</sup> interaction only in response to inducers
of cytoplasmic stress suggests that P52<sup>rIPK</sup> regulation of
P58<sup>IPK</sup> is specific to cytoplasmic stress-response pathways.
</p></sec><sec id="d7e2035"><title>Discussion</title><p><italic toggle="yes">The P52<sup>rIPK</sup></italic><sup></sup><italic toggle="yes"> Charged Domain and Regulation of P58<sup>IPK</sup></italic><sup></sup><italic toggle="yes">.</italic>
It has become increasingly clear that TPR proteins participate
in a diverse range of signaling processes, including protein
kinase regulation and stress-signaling cascades (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00044" ref-type="bibr"></xref>−<xref rid="bi020397eb00045" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi020397eb00046" ref-type="bibr"></xref></named-content></italic>).
Here we have shown that P52<sup>rIPK</sup> binds to P58<sup>IPK</sup> and inhibits
its function in vivo in a TPR domain 7-dependent manner.
TPR domain 7 is separated from TPR domain 6, which
mediates binding to PKR, by only 12 aa. The close proximity
of binding sites for P52<sup>rIPK</sup> and PKR raises the possibility
that the binding of these proteins to P58<sup>IPK</sup> is mutually
exclusive. Thus, P52<sup>rIPK</sup> may inhibit the PKR regulatory
function of P58<sup>IPK</sup> by acting as a noncompetitive inhibitor,
binding P58<sup>IPK</sup> and excluding the PKR−P58<sup>IPK</sup> interaction.
</p><p>We have also shown that the 114 aa charged domain of
P52<sup>rIPK</sup> is necessary and sufficient for interaction with P58<sup>IPK</sup>
and that this region is required for the regulation of P58<sup>IPK</sup>
in vivo. This region of P52<sup>rIPK</sup> is similar to the charged
domains of Hsp90, which act as protein interaction motifs.
In particular, there is evidence that the C-terminal charged
domain of Hsp90 binds to the TPR proteins Hop, FKBP52,
FKBP51, Cyp40, and PP5 (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00047" ref-type="bibr"></xref>, <xref rid="bi020397eb00048" ref-type="bibr"></xref></named-content></italic>). When we used computer-assisted homology search programs to look for sequence
similarity between overlapping 5−10 aa regions of the
P52<sup>rIPK</sup> and Hsp90 charged domains, we found that both
proteins contained the 6 aa motif KRIKEL (corresponding
to aa 101−106 of P52<sup>rIPK</sup>). This motif, or a conserved variant,
is also present within the TPR-binding regions of other
cellular and viral proteins, including c-Myb (<italic toggle="yes"><xref rid="bi020397eb00049" ref-type="bibr"></xref></italic>), human
immunodeficiency virus Vpu and Gag (<italic toggle="yes"><xref rid="bi020397eb00050" ref-type="bibr"></xref></italic>), and human and
murine PKR (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00024" ref-type="bibr"></xref>, <xref rid="bi020397eb00025" ref-type="bibr"></xref></named-content></italic>). The presence of this motif within a
diverse group of cellular and viral TPR-binding proteins
suggests that it may participate in binding to the TPR domain.
Indeed, mutations that localize very near and around this
motif within the C terminus of Hsp90 preclude its ability to
interact with PP5, FKBP52, and Hop (<italic toggle="yes"><xref rid="bi020397eb00038" ref-type="bibr"></xref></italic>).
</p><p><italic toggle="yes">A Role for P52<sup>rIPK</sup></italic><sup></sup><italic toggle="yes"> Regulation of P58<sup>IPK</sup></italic><sup></sup><italic toggle="yes"> in Stress-Induced
Signaling and Cell Growth Control.</italic> Serum withdrawal, heat
shock, and sodium arsenite treatment all induce the activation
and regulation of PKR (<italic toggle="yes"><xref rid="bi020397eb00001" ref-type="bibr"></xref></italic>). Each of these regimens also
induced the formation of a recoverable P52<sup>rIPK</sup>−P58<sup>IPK</sup>
complex. Importantly, these treatments all induce cytoplasmic
stress-response programs, either through serum-responsive
signaling cascades and the direct accumulation of heat shock
proteins or, in the case of sodium arsenite, by inducing the
inactivation of protein sulfhydryl groups (<italic toggle="yes"><xref rid="bi020397eb00001" ref-type="bibr"></xref></italic>). In contrast,
thapsigargin or tunicamycin treatment, which indirectly
activates PERK by inducing ER stress through the depletion
of ER calcium stores or inhibition of N-linked glycosylation,
respectively (<italic toggle="yes"><xref rid="bi020397eb00015" ref-type="bibr"></xref></italic>), did not induce discernible P52<sup>rIPK</sup>−P58<sup>IPK</sup>
complex formation. These results indicate that P52<sup>rIPK</sup> likely
mediates control of P58<sup>IPK</sup> function and PKR activity in
response to cytoplasmic stress signaling. Although we cannot
formally exclude a role for P52<sup>rIPK</sup> in the response to ER
stress, our results imply that P52<sup>rIPK</sup> regulates P58<sup>IPK</sup> in stress-response pathways that are functionally distinct from those
that impact the activity of PERK. This is particularly
intriguing since P58<sup>IPK</sup> expression is induced during ER stress
(<italic toggle="yes"><xref rid="bi020397eb00051" ref-type="bibr"></xref></italic>), and the lack of association with P52<sup>rIPK</sup> may correlate
with a need for increased P58<sup>IPK</sup> function. In this regard, we
have recently found that P58<sup>IPK</sup> interacts with PERK in ER-stressed cells, suggesting that P58<sup>IPK</sup> may also function as a
regulator of PERK activity (unpublished data).
</p><p>It is notable that PACT, a protein activator of PKR, is
also signaled to bind and activate the kinase in response to
a diverse range of stress signals, including serum deprivation
(<italic toggle="yes"><xref rid="bi020397eb00052" ref-type="bibr"></xref></italic>). In response to cellular stress, PACT stimulates the PKR-dependent phosphorylation of eIF2α, which correlates with
PACT phosphorylation. This suggests that PACT is signaled
to specifically activate PKR during the cellular stress
response. The observation that PACT and P52<sup>rIPK</sup> bind PKR
and P58<sup>IPK</sup>, respectively, in response to serum withdrawal
suggests that these proteins participate in a common pathway.
Thus, the binding of P52<sup>rIPK</sup> to P58<sup>IPK</sup>, and the inhibition of
P58<sup>IPK</sup> function, may potentiate PACT-mediated activation
of PKR in response to stress-induced signals. This may
provide the cell a rapid mechanism by which to downmodulate gene expression during the stress response.
</p><p>The cellular response to stress is clearly dependent upon
the nature and duration of the stress-induced signals. We
previously demonstrated that when cells are subjected to heat
shock, the association between P58<sup>IPK</sup> and Hsp40 is disrupted
during the recovery phase (<italic toggle="yes"><xref rid="bi020397eb00023" ref-type="bibr"></xref></italic>). However, the role of P52<sup>rIPK</sup>
in this process was not addressed. In the present study, we
demonstrated the formation of a P52<sup>rPK</sup>−P58<sup>IPK</sup> complex in
heat-shocked cells. It is possible that Hsp40 and P52<sup>rIPK</sup>
respond to different signals to mediate regulation of P58<sup>IPK</sup>
at different times and under different circumstances. It will
therefore be important to elucidate the signaling mechanisms
that lead to activation of P52<sup>rIPK</sup>. In this regard, preliminary
studies from our laboratory suggest that P52<sup>rIPK</sup> is phosphorylated during the stress response induced by serum withdrawal (unpublished data).
</p><p>Finally, through its ability to inhibit P58<sup>IPK</sup>, P52<sup>rIPK</sup> may
participate in cell growth regulation by acting as an upstream
modulator of both PKR-dependent and independent events.
Expression of trans-dominant negative PKR mutants induces
oncogenic transformation of murine fibroblast cell lines,
presumably by disrupting PKR-dependent processes of
translational control and apoptotic signaling (<italic toggle="yes"><named-content content-type="bibref-group"><xref rid="bi020397eb00053" ref-type="bibr"></xref>−<xref rid="bi020397eb00054" specific-use="suppress-in-print" ref-type="bibr"></xref><xref rid="bi020397eb00055" ref-type="bibr"></xref></named-content></italic>). Similarly, the overexpression of cellular and viral inhibitors of
PKR, including P58<sup>IPK</sup>, induces a transformed phenotype in
NIH3T3 cells (<italic toggle="yes">28</italic>, <italic toggle="yes">56</italic>, <italic toggle="yes">57</italic>). We also demonstrated that P58<sup>IPK</sup>
possesses oncogenic properties that are independent of PKR,
since the overexpression of P58<sup>IPK</sup> ΔTPR6, which fails to
interact with or inhibit PKR, also induces a transformed
phenotype (<italic toggle="yes"><xref rid="bi020397eb00029" ref-type="bibr"></xref></italic>). Taken together, these studies indicate that
regulation of PKR and P58<sup>IPK</sup> function is critical for
maintaining control of cell growth and proliferation. Thus,
P52<sup>rIPK</sup> may itself possess antiproliferative properties by
modulating the oncogenic potential of P58<sup>IPK</sup>.
</p></sec></body><back><ack><title>Acknowledgments</title><p>We thank Marlene Wambach and Cecelia Boyer for
excellent technical assistance and Drs. Ara Hovanessian and
Thomas Dever for anti-PKR 71/10 Mab and rabbit antiserum
to yeast eIF2α, respectively. We also thank Michael Bates
for assistance in figure preparation and Dr. Brenda Fredericksen for critical review of the manuscript.
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Tel: 206-732-6136.Fax: 206-732-6056. E-mail: honey@u.washington.edu.</affiliation><role><roleTerm type="text">author</roleTerm></role></name><typeOfResource>text</typeOfResource><genre type="research-article" displayLabel="research-article" authority="ISTEX" authorityURI="https://content-type.data.istex.fr" valueURI="https://content-type.data.istex.fr/ark:/67375/XTP-1JC4F85T-7">research-article</genre><originInfo><publisher>American Chemical Society</publisher><dateCreated encoding="w3cdtf">2002-08-20</dateCreated><dateIssued encoding="w3cdtf">2002-10-01</dateIssued><copyrightDate encoding="w3cdtf">2002</copyrightDate></originInfo><note type="footnote" ID="bi020397eAF2"> Financial support was provided by National Institutes of Health Grants AI22646 (to M.G.K.) and AI48235 (to M.G.). M.G. also received support from the Helen Hay Whitney Foundation and the State of Texas Applied Research Program (no. 0138-1999).</note><language><languageTerm type="code" authority="iso639-2b">eng</languageTerm><languageTerm type="code" authority="rfc3066">en</languageTerm></language><abstract>The 52 kDa protein referred to as P52rIPK was first identified as a regulator of P58IPK, a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52rIPK and P58IPK each possess structural domains implicated in stress signaling, including the charged domain of P52rIPK and the tetratricopeptide repeat (TPR) and DnaJ domains of P58IPK. The P52rIPK charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52rIPK function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58IPK. This domain bound specifically to P58IPK TPR domain 7, the domain adjacent to the TPR motif required for P58IPK interaction with PKR, thus providing a mechanism for P52rIPK inhibition of P58IPK function. Both the charged domain of P52rIPK and the TPR 7 domain of P58IPK were required for P52rIPK to mediate downstream control of PKR activity, eIF2α phosphorylation, and cell growth. Furthermore, we found that P52rIPK and P58IPK formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52rIPK charged domain functions as a TPR-specific signaling motif to directly regulate P58IPK within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.</abstract><note type="footnote" ID="bi020397eAF2"> Financial support was provided by National Institutes of Health Grants AI22646 (to M.G.K.) and AI48235 (to M.G.). 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