Cks1 proteasomal turnover is a predominant mode of regulation in breast cancer cells: Role of key tyrosines and lysines
Constitutive levels of Cks1 protein are very high in mammary carcinoma tissue and in breast tumor cell lines. However, despite being transcribed at relatively high levels, Cks1 protein is very low in normal mammary tissue. Also, basal Cks1 is barely detectable in primary human mammary epithelial cel...
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description | Constitutive levels of Cks1 protein are very high in mammary carcinoma tissue and in breast tumor cell lines. However, despite being transcribed at relatively high levels, Cks1 protein is very low in normal mammary tissue. Also, basal Cks1 is barely detectable in primary human mammary epithelial cells (HMECs). Epoximicin, a proteasome inhibitor, induced detectable endogenous Cks1 in HMECs, and upregulated it above the basal level in MCF-7 breast cancer cells. Interestingly, transiently transfected Cks1 is remarkably unstable and accumulates only upon proteasomal blockade in multiple cell lines even when driven by the strong CMV promoter-enhancer. We examined the stability of site-directed Cks1 mutants in order to identify the structural determinants of its turnover in cancer cells. Since protein turnover is regulated by phosphorylation, and phosphoproteomic studies reveal phosphorylated tyrosines in Cks1, we replaced its five conserved tyrosines (Y) with phenylalanine (F), both individually and in combinations. We find that like wild-type, all transiently transfected mutant Cks1 vectors, even when driven by the CMV promoter-enhancer, expressed detectable protein only in cells treated with epoximicin. However, turnover of the Y8F, Y12F and Y19F Cks1 mutants was more rapid than that of wild-type, Y7F and Y57F. Since lysines are modified by ubiquitination or acetylation we also examined the consequences of lysine to arginine (K-R) substitutions on Cks1 proteasomal turnover. We found that the individual mutations K4R, K26R, K30R, and K34R slowed Cks1 turnover, while the K79R mutation or the combined mutation K75-76-78-79R increased turnover. Taken together, regulation of Cks1 protein stability is crucially dependent on specific tyrosine and lysine residues which are potential sites for post-translational modifications. |
doi_str_mv | 10.3892/ijo.2014.2728 |
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However, despite being transcribed at relatively high levels, Cks1 protein is very low in normal mammary tissue. Also, basal Cks1 is barely detectable in primary human mammary epithelial cells (HMECs). Epoximicin, a proteasome inhibitor, induced detectable endogenous Cks1 in HMECs, and upregulated it above the basal level in MCF-7 breast cancer cells. Interestingly, transiently transfected Cks1 is remarkably unstable and accumulates only upon proteasomal blockade in multiple cell lines even when driven by the strong CMV promoter-enhancer. We examined the stability of site-directed Cks1 mutants in order to identify the structural determinants of its turnover in cancer cells. Since protein turnover is regulated by phosphorylation, and phosphoproteomic studies reveal phosphorylated tyrosines in Cks1, we replaced its five conserved tyrosines (Y) with phenylalanine (F), both individually and in combinations. We find that like wild-type, all transiently transfected mutant Cks1 vectors, even when driven by the CMV promoter-enhancer, expressed detectable protein only in cells treated with epoximicin. However, turnover of the Y8F, Y12F and Y19F Cks1 mutants was more rapid than that of wild-type, Y7F and Y57F. Since lysines are modified by ubiquitination or acetylation we also examined the consequences of lysine to arginine (K-R) substitutions on Cks1 proteasomal turnover. We found that the individual mutations K4R, K26R, K30R, and K34R slowed Cks1 turnover, while the K79R mutation or the combined mutation K75-76-78-79R increased turnover. Taken together, regulation of Cks1 protein stability is crucially dependent on specific tyrosine and lysine residues which are potential sites for post-translational modifications.</description><identifier>ISSN: 1019-6439</identifier><identifier>EISSN: 1791-2423</identifier><identifier>DOI: 10.3892/ijo.2014.2728</identifier><identifier>PMID: 25353373</identifier><language>eng</language><publisher>Greece: D.A. Spandidos</publisher><subject>Breast cancer ; Breast Neoplasms - metabolism ; Breast Neoplasms - pathology ; Cancer ; Carcinogens ; CDC2-CDC28 Kinases - chemistry ; CDC2-CDC28 Kinases - genetics ; CDC2-CDC28 Kinases - metabolism ; Cell cycle ; Cells, Cultured ; Cks1 ; Cks2 ; Dehydrogenases ; Development and progression ; Epidermal growth factor ; Female ; Gene expression ; Gene mutations ; Genetic aspects ; HEK293 Cells ; HeLa Cells ; Humans ; Kinases ; Laboratories ; Lysine - genetics ; Lysine - metabolism ; MCF-7 Cells ; Mutagenesis, Site-Directed ; p130/Rb2 ; p27Kip1 ; Phosphorylation ; Proteasome Endopeptidase Complex - metabolism ; Protein biosynthesis ; Protein Interaction Domains and Motifs - genetics ; Protein Processing, Post-Translational ; Proteins ; Proteolysis ; SCF ubiquitin ligase, proteasome ; Skp2 ; Studies ; Tyrosine - genetics ; Tyrosine - metabolism</subject><ispartof>International journal of oncology, 2015-01, Vol.46 (1), p.395-406</ispartof><rights>Copyright © 2015, Spandidos Publications</rights><rights>COPYRIGHT 2015 Spandidos Publications</rights><rights>Copyright Spandidos Publications UK Ltd. 2015</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c490t-564f90a4969c1984eaa470c5f0c673f4f8045981d606fd96488d9990ac7653613</citedby><cites>FETCH-LOGICAL-c490t-564f90a4969c1984eaa470c5f0c673f4f8045981d606fd96488d9990ac7653613</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,780,784,5571,27924,27925</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/25353373$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>KHATTAR, VINAYAK</creatorcontrib><creatorcontrib>THOTTASSERY, JAIDEEP V</creatorcontrib><title>Cks1 proteasomal turnover is a predominant mode of regulation in breast cancer cells: Role of key tyrosines and lysines</title><title>International journal of oncology</title><addtitle>Int J Oncol</addtitle><description>Constitutive levels of Cks1 protein are very high in mammary carcinoma tissue and in breast tumor cell lines. However, despite being transcribed at relatively high levels, Cks1 protein is very low in normal mammary tissue. Also, basal Cks1 is barely detectable in primary human mammary epithelial cells (HMECs). Epoximicin, a proteasome inhibitor, induced detectable endogenous Cks1 in HMECs, and upregulated it above the basal level in MCF-7 breast cancer cells. Interestingly, transiently transfected Cks1 is remarkably unstable and accumulates only upon proteasomal blockade in multiple cell lines even when driven by the strong CMV promoter-enhancer. We examined the stability of site-directed Cks1 mutants in order to identify the structural determinants of its turnover in cancer cells. Since protein turnover is regulated by phosphorylation, and phosphoproteomic studies reveal phosphorylated tyrosines in Cks1, we replaced its five conserved tyrosines (Y) with phenylalanine (F), both individually and in combinations. We find that like wild-type, all transiently transfected mutant Cks1 vectors, even when driven by the CMV promoter-enhancer, expressed detectable protein only in cells treated with epoximicin. However, turnover of the Y8F, Y12F and Y19F Cks1 mutants was more rapid than that of wild-type, Y7F and Y57F. Since lysines are modified by ubiquitination or acetylation we also examined the consequences of lysine to arginine (K-R) substitutions on Cks1 proteasomal turnover. We found that the individual mutations K4R, K26R, K30R, and K34R slowed Cks1 turnover, while the K79R mutation or the combined mutation K75-76-78-79R increased turnover. Taken together, regulation of Cks1 protein stability is crucially dependent on specific tyrosine and lysine residues which are potential sites for post-translational modifications.</description><subject>Breast cancer</subject><subject>Breast Neoplasms - metabolism</subject><subject>Breast Neoplasms - pathology</subject><subject>Cancer</subject><subject>Carcinogens</subject><subject>CDC2-CDC28 Kinases - chemistry</subject><subject>CDC2-CDC28 Kinases - genetics</subject><subject>CDC2-CDC28 Kinases - metabolism</subject><subject>Cell cycle</subject><subject>Cells, Cultured</subject><subject>Cks1</subject><subject>Cks2</subject><subject>Dehydrogenases</subject><subject>Development and progression</subject><subject>Epidermal growth factor</subject><subject>Female</subject><subject>Gene expression</subject><subject>Gene mutations</subject><subject>Genetic aspects</subject><subject>HEK293 Cells</subject><subject>HeLa Cells</subject><subject>Humans</subject><subject>Kinases</subject><subject>Laboratories</subject><subject>Lysine - genetics</subject><subject>Lysine - metabolism</subject><subject>MCF-7 Cells</subject><subject>Mutagenesis, Site-Directed</subject><subject>p130/Rb2</subject><subject>p27Kip1</subject><subject>Phosphorylation</subject><subject>Proteasome Endopeptidase Complex - metabolism</subject><subject>Protein biosynthesis</subject><subject>Protein Interaction Domains and Motifs - genetics</subject><subject>Protein Processing, Post-Translational</subject><subject>Proteins</subject><subject>Proteolysis</subject><subject>SCF ubiquitin ligase, proteasome</subject><subject>Skp2</subject><subject>Studies</subject><subject>Tyrosine - genetics</subject><subject>Tyrosine - metabolism</subject><issn>1019-6439</issn><issn>1791-2423</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2015</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><sourceid>ABUWG</sourceid><sourceid>AFKRA</sourceid><sourceid>BENPR</sourceid><sourceid>CCPQU</sourceid><recordid>eNptkd1rFDEUxQdRbK0--ioBwT7Nmkw-ZuJbWawKBUH0OaT5aLPNJGuSqex_791urRYkD7kkv3Mv95yue03wik5yeB82eTVgwlbDOExPumMyStIPbKBPocZE9oJRedS9qHWD8cA5Js-7o4FTTulIj7tf65tK0Lbk5nTNs46oLSXlW1dQqEjDj7N5DkmnhuZsHcoeFXe1RN1CTigkdFlA2ZDRyYDIuBjrB_Qtxzv0xu1Q25VcQ3LQLlkUd3f1y-6Z17G6V_f3Sffj_OP39ef-4uunL-uzi94wiVvPBfMSayaFNEROzGnNRmy4x0aM1DM_YcblRKzAwlsp2DRZKUFhRsGpIPSke3voCyv-XFxtapNhQRipiKQDJZRK-pe60tGpkHxuRZs5VKPOGCZ0wmAcUKv_UHCsm4PJyfkA748E7_4RXDsd23XNcdlbVx-D_QE0YFUtzqttCbMuO0Ww2sesIGa1j1ntYwb-zf1Wy-Xs7AP9J1cATg9A3YLpweb6wECnnokekx5TyelvXpetTg</recordid><startdate>201501</startdate><enddate>201501</enddate><creator>KHATTAR, VINAYAK</creator><creator>THOTTASSERY, JAIDEEP V</creator><general>D.A. 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metabolism</topic><topic>Breast Neoplasms - pathology</topic><topic>Cancer</topic><topic>Carcinogens</topic><topic>CDC2-CDC28 Kinases - chemistry</topic><topic>CDC2-CDC28 Kinases - genetics</topic><topic>CDC2-CDC28 Kinases - metabolism</topic><topic>Cell cycle</topic><topic>Cells, Cultured</topic><topic>Cks1</topic><topic>Cks2</topic><topic>Dehydrogenases</topic><topic>Development and progression</topic><topic>Epidermal growth factor</topic><topic>Female</topic><topic>Gene expression</topic><topic>Gene mutations</topic><topic>Genetic aspects</topic><topic>HEK293 Cells</topic><topic>HeLa Cells</topic><topic>Humans</topic><topic>Kinases</topic><topic>Laboratories</topic><topic>Lysine - genetics</topic><topic>Lysine - metabolism</topic><topic>MCF-7 Cells</topic><topic>Mutagenesis, Site-Directed</topic><topic>p130/Rb2</topic><topic>p27Kip1</topic><topic>Phosphorylation</topic><topic>Proteasome Endopeptidase Complex - metabolism</topic><topic>Protein biosynthesis</topic><topic>Protein Interaction Domains and Motifs - genetics</topic><topic>Protein Processing, Post-Translational</topic><topic>Proteins</topic><topic>Proteolysis</topic><topic>SCF ubiquitin ligase, proteasome</topic><topic>Skp2</topic><topic>Studies</topic><topic>Tyrosine - genetics</topic><topic>Tyrosine - metabolism</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>KHATTAR, VINAYAK</creatorcontrib><creatorcontrib>THOTTASSERY, JAIDEEP V</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>ProQuest Central (Corporate)</collection><collection>Nursing & Allied Health Database</collection><collection>Health & Medical Collection</collection><collection>ProQuest Central (purchase pre-March 2016)</collection><collection>Medical Database (Alumni Edition)</collection><collection>Hospital Premium Collection</collection><collection>Hospital Premium Collection (Alumni Edition)</collection><collection>ProQuest Central (Alumni) (purchase pre-March 2016)</collection><collection>ProQuest Central (Alumni Edition)</collection><collection>ProQuest Central UK/Ireland</collection><collection>British Nursing Database</collection><collection>ProQuest Central</collection><collection>ProQuest One Community College</collection><collection>Health Research Premium Collection</collection><collection>Health Research Premium Collection (Alumni)</collection><collection>ProQuest Health & Medical Complete (Alumni)</collection><collection>Nursing & Allied Health Database (Alumni Edition)</collection><collection>Health & Medical Collection (Alumni Edition)</collection><collection>Medical Database</collection><collection>Nursing & Allied Health Premium</collection><collection>ProQuest One Academic Eastern Edition (DO NOT USE)</collection><collection>ProQuest One Academic</collection><collection>ProQuest One Academic UKI Edition</collection><collection>ProQuest Central China</collection><jtitle>International journal of oncology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>KHATTAR, VINAYAK</au><au>THOTTASSERY, JAIDEEP V</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Cks1 proteasomal turnover is a predominant mode of regulation in breast cancer cells: Role of key tyrosines and lysines</atitle><jtitle>International journal of oncology</jtitle><addtitle>Int J Oncol</addtitle><date>2015-01</date><risdate>2015</risdate><volume>46</volume><issue>1</issue><spage>395</spage><epage>406</epage><pages>395-406</pages><issn>1019-6439</issn><eissn>1791-2423</eissn><abstract>Constitutive levels of Cks1 protein are very high in mammary carcinoma tissue and in breast tumor cell lines. However, despite being transcribed at relatively high levels, Cks1 protein is very low in normal mammary tissue. Also, basal Cks1 is barely detectable in primary human mammary epithelial cells (HMECs). Epoximicin, a proteasome inhibitor, induced detectable endogenous Cks1 in HMECs, and upregulated it above the basal level in MCF-7 breast cancer cells. Interestingly, transiently transfected Cks1 is remarkably unstable and accumulates only upon proteasomal blockade in multiple cell lines even when driven by the strong CMV promoter-enhancer. We examined the stability of site-directed Cks1 mutants in order to identify the structural determinants of its turnover in cancer cells. Since protein turnover is regulated by phosphorylation, and phosphoproteomic studies reveal phosphorylated tyrosines in Cks1, we replaced its five conserved tyrosines (Y) with phenylalanine (F), both individually and in combinations. We find that like wild-type, all transiently transfected mutant Cks1 vectors, even when driven by the CMV promoter-enhancer, expressed detectable protein only in cells treated with epoximicin. However, turnover of the Y8F, Y12F and Y19F Cks1 mutants was more rapid than that of wild-type, Y7F and Y57F. Since lysines are modified by ubiquitination or acetylation we also examined the consequences of lysine to arginine (K-R) substitutions on Cks1 proteasomal turnover. We found that the individual mutations K4R, K26R, K30R, and K34R slowed Cks1 turnover, while the K79R mutation or the combined mutation K75-76-78-79R increased turnover. Taken together, regulation of Cks1 protein stability is crucially dependent on specific tyrosine and lysine residues which are potential sites for post-translational modifications.</abstract><cop>Greece</cop><pub>D.A. Spandidos</pub><pmid>25353373</pmid><doi>10.3892/ijo.2014.2728</doi><tpages>12</tpages><oa>free_for_read</oa></addata></record> |
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source | Spandidos Publications Journals; MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
subjects | Breast cancer Breast Neoplasms - metabolism Breast Neoplasms - pathology Cancer Carcinogens CDC2-CDC28 Kinases - chemistry CDC2-CDC28 Kinases - genetics CDC2-CDC28 Kinases - metabolism Cell cycle Cells, Cultured Cks1 Cks2 Dehydrogenases Development and progression Epidermal growth factor Female Gene expression Gene mutations Genetic aspects HEK293 Cells HeLa Cells Humans Kinases Laboratories Lysine - genetics Lysine - metabolism MCF-7 Cells Mutagenesis, Site-Directed p130/Rb2 p27Kip1 Phosphorylation Proteasome Endopeptidase Complex - metabolism Protein biosynthesis Protein Interaction Domains and Motifs - genetics Protein Processing, Post-Translational Proteins Proteolysis SCF ubiquitin ligase, proteasome Skp2 Studies Tyrosine - genetics Tyrosine - metabolism |
title | Cks1 proteasomal turnover is a predominant mode of regulation in breast cancer cells: Role of key tyrosines and lysines |
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