Cks1 proteasomal turnover is a predominant mode of regulation in breast cancer cells: Role of key tyrosines and lysines

Khattar,Vinayak; Thottassery,Jaideep V.

doi:10.3892/ijo.2014.2728

Cks1 proteasomal turnover is a predominant mode of regulation in breast cancer cells: Role of key tyrosines and lysines

Authors:
- Vinayak Khattar
- Jaideep V. Thottassery
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Affiliations: Southern Research Institute, Birmingham, AL, USA
Published online on: October 23, 2014 https://doi.org/10.3892/ijo.2014.2728
Pages: 395-406

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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.

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1	Khattar V and Thottassery JV: Cks1: structure, emerging roles and implications in multiple cancers. J Cancer Ther. 4:1341–1354. 2013. View Article : Google Scholar : PubMed/NCBI
2	Ganoth D, Bornstein G, Ko TK, Larsen B, Tyers M, Pagano M and Hershko A: The cell-cycle regulatory protein Cks1 is required for SCF(Skp2)-mediated ubiquitinylation of p27. Nat Cell Biol. 3:321–324. 2001. View Article : Google Scholar : PubMed/NCBI
3	Spruck C, Strohmaier H, Watson M, et al: A CDK-independent function of mammalian Cks1: targeting of SCF(Skp2) to the CDK inhibitor p27^Kip1. Mol Cell. 7:639–650. 2001. View Article : Google Scholar : PubMed/NCBI
4	Westbrook L, Manuvakhova M, Kern FG, Estes NR, Ramanathan HN and Thottassery JV: Cks1 regulates cdk1 expression: a novel role during mitotic entry in breast cancer cells. Cancer Res. 67:11393–11401. 2007. View Article : Google Scholar : PubMed/NCBI
5	Hoellein A, Graf S, Bassermann F, et al: Cks1 promotion of S phase entry and proliferation is independent of p27^Kip1 suppression. Mol Cell Biol. 32:2416–2427. 2012. View Article : Google Scholar : PubMed/NCBI
6	Liberal V, Martinsson-Ahlzen HS, Liberal J, Spruck CH, Widschwendter M, McGowan CH and Reed SI: Cyclin-dependent kinase subunit (Cks) 1 or Cks2 overexpression overrides the DNA damage response barrier triggered by activated oncoproteins. Proc Natl Acad Sci USA. 109:2754–2759. 2012. View Article : Google Scholar : PubMed/NCBI
7	Westbrook L, Ramanathan HN, Isayeva T, et al: High Cks1 expression in transgenic and carcinogen-initiated mammary tumors is not always accompanied by reduction in p27^Kip1. Int J Oncol. 34:1425–1431. 2009.PubMed/NCBI
8	Slotky M, Shapira M, Ben-Izhak O, Linn S, Futerman B, Tsalic M and Hershko DD: The expression of the ubiquitin ligase subunit Cks1 in human breast cancer. Breast Cancer Res. 7:R737–R744. 2005. View Article : Google Scholar : PubMed/NCBI
9	Shapira M, Ben-Izhak O, Slotky M, Goldin O, Lahav-Baratz S and Hershko DD: Expression of the ubiquitin ligase subunit cyclin kinase subunit 1 and its relationship to S-phase kinase protein 2 and p27^Kip1 in prostate cancer. J Urol. 176:2285–2289. 2006. View Article : Google Scholar : PubMed/NCBI
10	Lee SW, Kang SB, Lee DS and Lee JU: Akt and Cks1 are related with lymph node metastasis in gastric adenocarcinoma. Hepatogastroenterology. 60:932–937. 2013.PubMed/NCBI
11	Nagler RM, Ben-Izhak O, Ostrovsky D, Golz A and Hershko DD: The expression and prognostic significance of Cks1 in salivary cancer. Cancer Invest. 27:512–520. 2009. View Article : Google Scholar : PubMed/NCBI
12	Calvisi DF, Ladu S, Pinna F, et al: SKP2 and CKS1 promote degradation of cell cycle regulators and are associated with hepatocellular carcinoma prognosis. Gastroenterology. 137:1816–1826. 2009. View Article : Google Scholar : PubMed/NCBI
13	Chang H, Jiang N, Jiang H, Saha MN, Qi C, Xu W and Reece D: CKS1B nuclear expression is inversely correlated with p27^Kip1 expression and is predictive of an adverse survival in patients with multiple myeloma. Haematologica. 95:1542–1547. 2010. View Article : Google Scholar : PubMed/NCBI
14	Pardo I, Lillemoe HA, Blosser RJ, et al: Next-generation transcriptome sequencing of the premenopausal breast epithelium using specimens from a normal human breast tissue bank. Breast Cancer Res. 16:R262014. View Article : Google Scholar
15	Hershko A and Ciechanover A: The ubiquitin system for protein degradation. Annu Rev Biochem. 61:761–807. 1992. View Article : Google Scholar
16	Hattori T, Kitagawa K, Uchida C, Oda T and Kitagawa M: Cks1 is degraded via the ubiquitin-proteasome pathway in a cell cycle-dependent manner. Genes Cells. 8:889–896. 2003. View Article : Google Scholar : PubMed/NCBI
17	Bashir T, Dorrello NV, Amador V, Guardavaccaro D and Pagano M: Control of the SCF (Skp2-Cks1) ubiquitin ligase by the APC/C (Cdh1) ubiquitin ligase. Nature. 428:190–193. 2004. View Article : Google Scholar : PubMed/NCBI
18	Hunter T: The age of crosstalk: phosphorylation, ubiquitination, and beyond. Mol Cell. 28:730–738. 2007. View Article : Google Scholar : PubMed/NCBI
19	Ungureanu D, Saharinen P, Junttila I, Hilton DJ and Silvennoinen O: Regulation of Jak2 through the ubiquitin-proteasome pathway involves phosphorylation of Jak2 on Y1007 and interaction with SOCS-1. Mol Cell Biol. 22:3316–3326. 2002. View Article : Google Scholar : PubMed/NCBI
20	Ali S, Nouhi Z, Chughtai N and Ali S: SHP-2 regulates SOCS-1-mediated Janus kinase-2 ubiquitination/degradation downstream of the prolactin receptor. J Biol Chem. 278:52021–52031. 2003. View Article : Google Scholar : PubMed/NCBI
21	He G, Zhang YW, Lee JH, et al: AMP-activated protein kinase induces p53 by phosphorylating MDMX and inhibiting its activity. Mol Cell Biol. 34:148–157. 2014. View Article : Google Scholar : PubMed/NCBI
22	Nalavadi VC, Muddashetty RS, Gross C and Bassell GJ: Dephosphorylation-induced ubiquitination and degradation of FMRP in dendrites: a role in immediate early mGluR-stimulated translation. J Neurosci. 32:2582–2587. 2012. View Article : Google Scholar : PubMed/NCBI
23	Wolf-Yadlin A, Kumar N, Zhang Y, et al: Effects of HER2 over-expression on cell signaling networks governing proliferation and migration. Mol Syst Biol. 2:542006. View Article : Google Scholar : PubMed/NCBI
24	Hornbeck PV, Kornhauser JM, Tkachev S, Zhang B, Skrzypek E, Murray B, Latham V and Sullivan M: PhosphoSitePlus: a comprehensive resource for investigating the structure and function of experimentally determined post-translational modifications in man and mouse. Nucleic Acids Res. 40:D261–D270. 2012.PubMed/NCBI
25	Wu F, Wang P, Young LC, Lai R and Li L: Proteome-wide identification of novel binding partners to the oncogenic fusion gene protein, NPM-ALK, using tandem affinity purification and mass spectrometry. Am J Pathol. 174:361–370. 2009. View Article : Google Scholar : PubMed/NCBI
26	Olsen JV, Vermeulen M, Santamaria A, et al: Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal. 3:ra32010. View Article : Google Scholar : PubMed/NCBI
27	Shirayama M, Soto MC, Ishidate T, et al: The conserved kinases CDK-1, GSK-3, KIN-19, and MBK-2 promote OMA-1 destruction to regulate the oocyte-to-embryo transition in C. elegans. Curr Biol. 16:47–55. 2006. View Article : Google Scholar : PubMed/NCBI
28	Arvai AS, Bourne Y, Hickey MJ and Tainer JA: Crystal structure of the human cell cycle protein CksHs1: single domain fold with similarity to kinase N-lobe domain. J Mol Biol. 249:835–842. 1995. View Article : Google Scholar : PubMed/NCBI
29	Choudhary C, Kumar C, Gnad F, et al: Lysine acetylation targets protein complexes and co-regulates major cellular functions. Science. 325:834–840. 2009. View Article : Google Scholar : PubMed/NCBI
30	Kim W, Bennett EJ, Huttlin EL, et al: Systematic and quantitative assessment of the ubiquitin-modified proteome. Mol Cell. 44:325–340. 2011. View Article : Google Scholar : PubMed/NCBI
31	Pfaffl MW: A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res. 29:e452001. View Article : Google Scholar : PubMed/NCBI
32	Holt LJ: Regulatory modules: coupling protein stability to phosphoregulation during cell division. FEBS Lett. 586:2773–2777. 2012. View Article : Google Scholar : PubMed/NCBI
33	Hu Y, Sun H, Drake J, et al: From mice to humans: identification of commonly deregulated genes in mammary cancer via comparative SAGE studies. Cancer Res. 64:7748–7755. 2004. View Article : Google Scholar : PubMed/NCBI
34	Kaneko T, Joshi R, Feller SM and Li SS: Phosphotyrosine recognition domains: the typical, the atypical and the versatile. Cell Commun Signal. 10:322012. View Article : Google Scholar : PubMed/NCBI
35	Burshtyn DN, Yang W, Yi T and Long EO: A novel phosphotyrosine motif with a critical amino acid at position -2 for the SH2 domain-mediated activation of the tyrosine phosphatase SHP-1. J Biol Chem. 272:13066–13072. 1997. View Article : Google Scholar : PubMed/NCBI
36	Songyang Z: Recognition and regulation of primary-sequence motifs by signaling modular domains. Prog Biophys Mol Biol. 71:359–372. 1999. View Article : Google Scholar : PubMed/NCBI
37	Heo WD, Inoue T, Park WS, Kim ML, Park BO, Wandless TJ and Meyer T: PI(3,4,5)P3 and PI(4,5)P2 lipids target proteins with polybasic clusters to the plasma membrane. Science. 314:1458–1461. 2006. View Article : Google Scholar : PubMed/NCBI