Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation
Main Article Content
Keywords
Cyclin-dependent kinase-1, Bcl-xL, caspase-9, peptide phosphorylation, peptide dephosphorylation, high resolution peptide gels
Abstract
Protein phosphorylation and dephosphorylation reactions play key regulatory roles in many fundamental cellular processes. Due to the large number of kinases and phosphatases in the genome, the identification of the specific enzymes responsible for a given site in a given protein is immensely challenging. However, because protein kinases and phosphatases recognize local specificity determinants within proteins, it is possible to use small peptides to study the characteristics of site-specific phosphorylation. In addition, phosphorylation usually causes retardation in gel mobility, providing an opportunity to investigate peptide phosphorylation and dephosphorylation by monitoring migration on high resolution peptide gels. In this study, we demonstrate the utility of such a technique using small peptides corresponding to cyclin-dependent kinase-1 (Cdk1)/cyclin B1 sites in two important apoptotic regulatory proteins, Bcl-xL and caspase-9. We show that the mobility of the peptides is retarded following Cdk1-mediated phosphorylation, and that peptide dephosphorylation, catalyzed either by purified phosphatase or by crude cell extracts, is readily observable by increased peptide gel mobility. Furthermore, the procedure can be conducted without the use of radioactive adenosine triphosphate (ATP), and does not require any specialized reagents or apparatus. The method can be used to identify and characterize specific kinase and phosphatases responsible for phosphorylation and dephosphorylation of specific sites in any protein of interest.
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References
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[2] C. Cai, L. Chen, X. Jiang, X. Lu, Modeling signal transduction from protein phosphorylation to gene expression, Cancer Inform 13 (2014) 59-67.
[3] C.S. Tan, Sequence, structure, and network evolution of protein phosphorylation, Sci Signal 4 (2011) mr6.
[4] J.A. Ubersax, J.E. Ferrell, Jr., Mechanisms of specificity in protein phosphorylation, Nat Rev Mol Cell Biol 8 (2007) 530-541.
[5] M.L. Miller, N. Blom, Kinase-specific prediction of protein phosphorylation sites, Methods Mol Biol 527 (2009) 299-310, x.
[6] A. Via, F. Diella, T.J. Gibson, M. Helmer-Citterich, From sequence to structural analysis in protein phosphorylation motifs, Front Biosci (Landmark Ed) 16 (2011) 1261-1275.
[7] M. Schutkowski, U. Reineke, U. Reimer, Peptide arrays for kinase profiling, Chembiochem 6 (2005) 513-521.
[8] D.T. Terrano, M. Upreti, T.C. Chambers, Cyclin-dependent kinase 1-mediated Bcl-xL/Bcl-2 phosphorylation acts as a functional link coupling mitotic arrest and apoptosis, Mol Cell Biol 30 (2010) 640-656.
[9] R. Chu, D.T. Terrano, T.C. Chambers, Cdk1/cyclin B plays a key role in mitotic arrest-induced apoptosis by phosphorylation of Mcl-1, promoting its degradation and freeing Bak from sequestration, Biochem Pharmacol 83 (2012) 199-206.
[10] N. Sakurikar, J.M. Eichhorn, T.C. Chambers, Cyclin-dependent kinase-1 (Cdk1)/cyclin B1 dictates cell fate after mitotic arrest via phosphoregulation of antiapoptotic Bcl-2 proteins, J Biol Chem 287 (2012) 39193-39204.
[11] J.M. Eichhorn, N. Sakurikar, S.E. Alford, R. Chu, T.C. Chambers, Critical role of anti-apoptotic Bcl-2 protein phosphorylation in mitotic death, Cell Death Dis 4 (2013) e834.
[12] E.A. Nigg, Mitotic kinases as regulators of cell division and its checkpoints, Nat Rev Mol Cell Biol 2 (2001) 21-32.
[13] J.V. Olsen, M. Vermeulen, A. Santamaria, C. Kumar, M.L. Miller, L.J. Jensen, F. Gnad, J. Cox, T.S. Jensen, E.A. Nigg, S. Brunak, M. Mann, Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis, Sci Signal 3 (2010) ra3.
[14] R.H. Medema, A. Lindqvist, Boosting and suppressing mitotic phosphorylation, Trends Biochem Sci 36 (2011) 578-584.
[15] N. Sakurikar, J.M. Eichhorn, S.E. Alford, T.C. Chambers, Identification of a mitotic death signature in cancer cell lines, Cancer Lett 343 (2014) 232-238.
[16] L.A. Allan, P.R. Clarke, Phosphorylation of caspase-9 by CDK1/cyclin B1 protects mitotic cells against apoptosis, Mol Cell 26 (2007) 301-310.
[17] P.R. Clarke, L.A. Allan, Cell-cycle control in the face of damage--a matter of life or death, Trends Cell Biol 19 (2009) 89-98.
[18] L.A. Allan, P.R. Clarke, Apoptosis and autophagy: Regulation of caspase-9 by phosphorylation, FEBS J 276 (2009) 6063-6073.
[19] M. Upreti, E.N. Galitovskaya, R. Chu, A.J. Tackett, D.T. Terrano, S. Granell, T.C. Chambers, Identification of the major phosphorylation site in Bcl-xL induced by microtubule inhibitors and analysis of its functional significance, J Biol Chem 283 (2008) 35517-35525.
[20] C. Salazar, T. Hofer, Multisite protein phosphorylation--from molecular mechanisms to kinetic models, FEBS J 276 (2009) 3177-3198.
[21] A. Basu, S. Haldar, Identification of a novel Bcl-xL phosphorylation site regulating the sensitivity of taxol- or 2-methoxyestradiol-induced apoptosis, FEBS Lett 538 (2003) 41-47.
[22] B.A. Weaver, D.W. Cleveland, Decoding the links between mitosis, cancer, and chemotherapy: The mitotic checkpoint, adaptation, and cell death, Cancer Cell 8 (2005) 7-12.
[23] C.H. Topham, S.S. Taylor, Mitosis and apoptosis: how is the balance set?, Curr Opin Cell Biol 25 (2013) 780-785.
[24] L. Du, C.S. Lyle, T.C. Chambers, Characterization of vinblastine-induced Bcl-xL and Bcl-2 phosphorylation: evidence for a novel protein kinase and a coordinated phosphorylation/dephosphorylation cycle associated with apoptosis induction, Oncogene 24 (2005) 107-117.
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Published
Aug 2, 2017
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Battle LJ, Chambers TC. Small peptide substrates and high resolution peptide gels for the analysis of site-specific protein phosphorylation and dephosphorylation. J Biol Methods [Internet]. 2017 Aug. 2 [cited 2024 Apr. 26];4(3):e76. Available from: https://jbmethods.org/index.php/jbm/article/view/199
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