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Cdc37 engages in stable, S14A mutation-reinforced association with the most atypical member of the yeast kinome, Cdk-activating kinase (Cak1)

Cell Stress and Chaperones volume 19, pages 695–703 (2014)Cite this article

Abstract

In most eukaryotes, Cdc37 is an essential chaperone, transiently associating with newly synthesised protein kinases in order to promote their stabilisation and activation. To determine whether the yeast Cdc37 participates in any stable protein interactions in vivo, genomic two-hybrid screens were conducted using baits that are functional as they preserve the integrity of the conserved N-terminal region of Cdc37, namely a Cdc37-Gal4 DNA binding domain (BD) fusion in both its wild type and its S14 nonphosphorylatable (Cdc37(S14A)) mutant forms. While this failed to identify the protein kinases previously identified as Cdc37 interactors in pull-down experiments, it did reveal Cdc37 engaging in a stable association with the most atypical member of the yeast kinome, cyclin-dependent kinase (Cdk1)-activating kinase (Cak1). Phosphorylation of the conserved S14 of Cdc37 is normally crucial for the interaction with, and stabilisation of, those protein kinase targets of Cdc37, Cak1 is unusual in that the lack of this Cdc37 S14 phosphorylation both reinforces Cak1:Cdc37 interaction and does not compromise Cak1 expression in vivo. Thus, this is the first Cdc37 client kinase found to be excluded from S14 phosphorylation-dependent interaction. The unusual stability of this Cak1:Cdc37 association may partly reflect unique structural features of the fungal Cak1.

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References

  • Abbas-Terki T, Donze O, Picard D (2000) The molecular chaperone Cdc37 is required for Ste11 function and pheromone-induced cell cycle arrest. FEBS Lett 467(1):111–116

    CAS  PubMed  Article  Google Scholar 

  • Bandhakavi S, McCann RO, Hanna DE, Glover CV (2003) A positive feedback loop between protein kinase CKII and Cdc37 promotes the activity of multiple protein kinases. J Biol Chem 278(5):2829–2836

    CAS  PubMed  Article  Google Scholar 

  • Bartel PL, Fields S (1997) The yeast two-hybrid system. Advances in molecular biology. Oxford University Press, New York

    Google Scholar 

  • Brugge JS, Erikson E, Erikson RL (1981) The specific interaction of the Rous sarcoma virus transforming protein, pp60src, with two cellular proteins. Cell 25(2):363–372

    CAS  PubMed  Article  Google Scholar 

  • Cross FR, Levine K (1998) Molecular evolution allows bypass of the requirement for activation loop phosphorylation of the Cdc28 cyclin-dependent kinase. Mol Cell Biol 18(5):2923–2931

    CAS  PubMed Central  PubMed  Google Scholar 

  • Enke DA, Kaldis P, Solomon MJ (2000) Kinetic analysis of the cyclin-dependent kinase-activating kinase (Cak1p) from budding yeast. J Biol Chem 275(43):33267–33271

    CAS  PubMed  Article  Google Scholar 

  • Farrell A, Morgan DO (2000) Cdc37 promotes the stability of protein kinases Cdc28 and Cak1. Mol Cell Biol 20(3):749–754

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Gavin AC, Bosche M, Krause R, Grandi P, Marzioch M, Bauer A, Schultz J, Rick JM, Michon AM, Cruciat CM, Remor M, Hofert C, Schelder M, Brajenovic M, Ruffner H, Merino A, Klein K, Hudak M, Dickson D, Rudi T, Gnau V, Bauch A, Bastuck S, Huhse B, Leutwein C, Heurtier MA, Copley RR, Edelmann A, Querfurth E, Rybin V, Drewes G, Raida M, Bouwmeester T, Bork P, Seraphin B, Kuster B, Neubauer G, Superti-Furga G (2002) Functional organization of the yeast proteome by systematic analysis of protein complexes. Nature 415(6868):141–147

    CAS  PubMed  Article  Google Scholar 

  • Geymonat M, Spanos A, Sedgwick SG (2007) A Saccharomyces cerevisiae autoselection system for optimised recombinant protein expression. Gene 399(2):120–128

    CAS  PubMed  Article  Google Scholar 

  • Hawle P, Horst D, Bebelman JP, Yang XX, Siderius M, van der Vies SM (2007) Cdc37p is required for stress-induced high-osmolarity glycerol and protein kinase C mitogen-activated protein kinase pathway functionality by interaction with Hog1p and Slt2p (Mpk1p). Eukaryot Cell 6(3):521–532

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • James P, Halladay J, Craig EA (1996) Genomic libraries and a host strain designed for highly efficient two-hybrid selection in yeast. Genetics 144(4):1425–1436

    CAS  PubMed Central  PubMed  Google Scholar 

  • Johnson JL, Brown C (2009) Plasticity of the Hsp90 chaperone machine in divergent eukaryotic organisms. Cell Stress Chaperones 14(1):83–94

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Kaldis P, Sutton A, Solomon MJ (1996) The Cdk-activating kinase (CAK) from budding yeast. Cell 86(4):553–564

    CAS  PubMed  Article  Google Scholar 

  • Kaldis P, Russo AA, Chou HS, Pavletich NP, Solomon MJ (1998) Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities. Mol Biol Cell 9(9):2545–2560

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Krogan NJ, Cagney G, Yu H, Zhong G, Guo X, Ignatchenko A, Li J, Pu S, Datta N, Tikuisis AP, Punna T, Peregrin-Alvarez JM, Shales M, Zhang X, Davey M, Robinson MD, Paccanaro A, Bray JE, Sheung A, Beattie B, Richards DP, Canadien V, Lalev A, Mena F, Wong P, Starostine A, Canete MM, Vlasblom J, Wu S, Orsi C, Collins SR, Chandran S, Haw R, Rilstone JJ, Gandi K, Thompson NJ, Musso G, St Onge P, Ghanny S, Lam MH, Butland G, Altaf-Ul AM, Kanaya S, Shilatifard A, O’Shea E, Weissman JS, Ingles CJ, Hughes TR, Parkinson J, Gerstein M, Wodak SJ, Emili A, Greenblatt JF (2006) Global landscape of protein complexes in the yeast Saccharomyces cerevisiae. Nature 440(7084):637–643. doi:10.1038/nature04670

    CAS  PubMed  Article  Google Scholar 

  • Lamphere L, Fiore F, Xu X, Brizuela L, Keezer S, Sardet C, Draetta GF, Gyuris J (1997) Interaction between Cdc37 and Cdk4 in human cells. Oncogene 14(16):1999–2004

    CAS  PubMed  Article  Google Scholar 

  • Longtine MS, McKenzie A 3rd, Demarini DJ, Shah NG, Wach A, Brachat A, Philippsen P, Pringle JR (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast (Chichester Engl) 14(10):953–961

    CAS  Article  Google Scholar 

  • MacLean M, Picard D (2003) Cdc37 goes beyond Hsp90 and kinases. Cell Stress Chaperones 8(2):114–119

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Mandal AK, Lee P, Chen JA, Nillegoda N, Heller A, Distasio S, Oen H, Victor J, Nair DM, Brodsky JL, Caplan AJ (2007) Cdc37 has distinct roles in protein kinase quality control that protect nascent chains from degradation and promote posttranslational maturation. J Cell Biol 176(3):319–328. doi:10.1083/jcb.200604106

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Millson SM, Truman A, Piper PW (2003) Vectors for N- or C-terminal positioning of the yeast Gal4p DNA binding or activator domains. Biotechniques 35:60–64

    CAS  PubMed  Google Scholar 

  • Millson SH, Truman A, Wolfram F, King V, Panaretou B, Prodromou C, Pearl LH, Piper PW (2004a) Investigating the protein-protein interactions of the yeast Hsp90 chaperone system by two hybrid analysis: potential uses and limitations of this approach. Cell Stress Chaperones 9:359–368

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Millson SH, Truman AW, Wolfram F, King V, Panaretou B, Prodromou C, Pearl LH, Piper PW (2004b) Investigating the protein-protein interactions of the yeast Hsp90 chaperone system by two-hybrid analysis: potential uses and limitations of this approach. Cell Stress Chaperones 9(4):359–368

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Millson SH, Truman A, King V, Prodromou C, Pearl L, Piper PW (2005a) A two-hybrid screen of the yeast proteome for Hsp90 interactors uncovers a novel Hsp90 chaperone requirement in activity of a stress-activated MAP kinase, Slt2p(Mpk1p). Eukaryot Cell 4:849–860. doi:10.1128/EC.4.5.849-860.2005

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Millson SH, Truman AW, King V, Prodromou C, Pearl LH, Piper PW (2005b) A two-hybrid screen of the yeast proteome for Hsp90 interactors uncovers a novel Hsp90 chaperone requirement in the activity of a stress-activated mitogen-activated protein kinase, Slt2p (Mpk1p). Eukaryot Cell 4(5):849–860. doi:10.1128/EC.4.5.849-860.2005

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Miyata Y, Nishida E (2004) CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37. Mol Cell Biol 24(9):4065–4074

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Mollapour M, Piper PW (2007) Hog1 mitogen-activated protein kinase phosphorylation targets the yeast Fps1 aquaglyceroporin for endocytosis, thereby rendering cells resistant to acetic acid. Mol Cell Biol 27:6446–6556

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Mort-Bontemps-Soret M, Facca C, Faye G (2002) Physical interaction of Cdc28 with Cdc37 in Saccharomyces cerevisiae. Mol Genet Genomics 267(4):447–458. doi:10.1007/s00438-002-0676-3

    CAS  PubMed  Article  Google Scholar 

  • Pearl LH (2005) Hsp90 and Cdc37 – a chaperone cancer conspiracy. Curr Opin Genet Dev 15(1):55–61

    CAS  PubMed  Article  Google Scholar 

  • Puig O, Caspary F, Rigaut G, Rutz B, Bouveret E, Bragado-Nilsson E, Wilm M, Seraphin B (2001) The tandem affinity purification (TAP) method: a general procedure of protein complex purification. Methods 24(3):218–229

    CAS  PubMed  Article  Google Scholar 

  • Rao J, Lee P, Benzeno S, Cardozo C, Albertus J, Robins DM, Caplan AJ (2001) Functional interaction of human Cdc37 with the androgen receptor but not with the glucocorticoid receptor. J Biol Chem 276(8):5814–5820. doi:10.1074/jbc.M007385200

    CAS  PubMed  Article  Google Scholar 

  • Roe SM, Ali MM, Meyer P, Vaughan CK, Panaretou B, Piper PW, Prodromou C, Pearl LH (2004) The mechanism of Hsp90 regulation by the protein kinase-specific cochaperone p50(cdc37). Cell 116(1):87–98

    CAS  PubMed  Article  Google Scholar 

  • Shao J, Irwin A, Hartson SD, Matts RL (2003) Functional dissection of cdc37: characterization of domain structure and amino acid residues critical for protein kinase binding. Biochemistry 42(43):12577–12588

    CAS  PubMed  Article  Google Scholar 

  • Siligardi G, Panaretou B, Meyer P, Singh S, Woolfson DN, Piper PW, Pearl LH, Prodromou C (2002) Regulation of Hsp90 ATPase activity by the co-chaperone Cdc37p/p50cdc37. J Biol Chem 277(23):20151–20159. doi:10.1074/jbc.M201287200

    CAS  PubMed  Article  Google Scholar 

  • Smith JR, Workman P (2009) Targeting CDC37: an alternative, kinase-directed strategy for disruption of oncogenic chaperoning. Cell Cycle (Georgetown Tex) 8(3):362–372

    CAS  Article  Google Scholar 

  • Song H, Hanlon N, Brown NR, Noble ME, Johnson LN, Barford D (2001) Phosphoprotein-protein interactions revealed by the crystal structure of kinase-associated phosphatase in complex with phosphoCDK2. Mol Cell 7(3):615–626

    CAS  PubMed  Article  Google Scholar 

  • Talevich E, Mirza A, Kannan N (2011) Structural and evolutionary divergence of eukaryotic protein kinases in Apicomplexa. BMC Evol Biol 11:321

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Tsakraklides V, Solomon MJ (2002) Comparison of Cak1p-like cyclin-dependent kinase-activating kinases. J Biol Chem 277(36):33482–33489

    CAS  PubMed  Article  Google Scholar 

  • Turnbull EL, Martin IV, Fantes PA (2005) Cdc37 maintains cellular viability in Schizosaccharomyces pombe independently of interactions with heat-shock protein 90. FEBS J 272(16):4129–4140

    CAS  PubMed  Article  Google Scholar 

  • Uetz P, Cagney G, Lockshon D, Qureshi-Emili A, Conover D, Johnston M, Fields S (2000) A protein array for genome-wide screens of protein-protein interactions. Nature 403:623–627

    CAS  PubMed  Article  Google Scholar 

  • Vaughan CK, Gohlke U, Sobott F, Good VM, Ali MM, Prodromou C, Robinson CV, Saibil HR, Pearl LH (2006) Structure of an Hsp90-Cdc37-Cdk4 complex. Mol Cell 23(5):697–707

    CAS  PubMed  Article  Google Scholar 

  • Vaughan CK, Mollapour M, Smith JR, Truman A, Hu B, Good VM, Panaretou B, Neckers L, Clarke PA, Workman P, Piper PW, Prodromou C, Pearl LH (2008) Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37. Mol Cell 31(6):886–895

    CAS  PubMed Central  PubMed  Article  Google Scholar 

  • Yang XX, Maurer KC, Molanus M, Mager WH, Siderius M, Vies SM (2006) The molecular chaperone Hsp90 is required for high osmotic stress response in Saccharomyces cerevisiae. FEMS Yeast Res 6(2):195–204

    CAS  PubMed  Article  Google Scholar 

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Acknowledgments

We are indebted to S. Lindquist, S. Fields, M. Geymonat and F. Cross for strains. This study was supported by The Netherlands Organisation for Scientific Research (project number 700.99.012) and the Federation of European Biochemical Societies (FEBS short-term fellowship to PH) and a Saudi Government Studentship (MAA).

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  1. Patricija van Oosten-Hawle

    Present address: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2205 Tech Drive, Hogan 2-100, Evanston, IL, 60208-3500, USA

Affiliations

  1. Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK

    Stefan Millson, Mohammed A. Alkuriji & Peter W. Piper

  2. Department of Biochemistry and Molecular Biology, Faculty of Science, VU University, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands

    Patricija van Oosten-Hawle

  3. University of Chicago, 929 E 57th Street, GCIS Room W519H, Chicago, IL, 60637, USA

    Andrew Truman

  4. Department of Medicinal Chemistry, Faculty of Science, VU University, De Boelelaan 1083, 1081 HV, Amsterdam, The Netherlands

    Marco Siderius

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  1. Stefan Millson
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Correspondence to Peter W. Piper.

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Millson, S., van Oosten-Hawle, P., Alkuriji, M.A. et al. Cdc37 engages in stable, S14A mutation-reinforced association with the most atypical member of the yeast kinome, Cdk-activating kinase (Cak1). Cell Stress and Chaperones 19, 695–703 (2014). https://doi.org/10.1007/s12192-014-0497-4

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  • DOI: https://doi.org/10.1007/s12192-014-0497-4

Keywords

  • Yeast two-hybrid
  • Cdc37
  • Molecular chaperone
  • Cak1