Biopolym. Cell. 2013; 29(2):136-142.
Molecular and Cell Biotechnologies
Generation of monoclonal antibody against protein phosphatase 5
1Malanchuk O. M., 1Ovcharenko G. V., 1Tykhonkova I. O., 2Khoma O. S., 2Chorna U. V., 2Rybak M. Yu., 1Palchevskyy S. S.
  1. Institute of Molecular Biology and Genetics, NAS of Ukraine
    150, Akademika Zabolotnoho Str., Kyiv, Ukraine, 03680
  2. Educational and Scientific Center "Institute of Biology",
    Taras Shevchenko National University of Kyiv
    64/13, Volodymyrska Str., Kyiv, Ukraine, 01601


Aim. Serine/threonine protein phosphatase 5 (PP5) is a unique member of serine/threonine phosphatase family, comprises a regulatory tetratricopeptide repeat (TPR) domain and regulates many cell signaling pathways. Here, we describe the development of a PP5 specific monoclonal antibody (mAb) and characterize its suitability for Western blotting and immunoprecipitation. Methods. Hybridoma technology has been used for monoclonal antibody generation. Immunization was carried out with recombinant mouse PP5 expressed in Escherichia coli as a GST-tagged fusion protein. Results. mAb against PP5 has been generated. Conclusions. Generated mAb specifically recognizes recombinant and endogenous mouse and rat PP5 and is suitable for Western blotting and immunoprecipitation. This mAb will be useful tool for investigations of PP5 physiological role.
Keywords: protein phosphatase 5, hybridoma technique, monoclonal antibody


[1] Russell L. C., Whitt S. R., Chen M. S., Chinkers M. Identification of conserved residues required for the binding of a tetratricopeptide repeat domain to heat shock protein 90 J. Biol. Chem 1999 274, N 29 P. 20060–20063.
[2] Chen M. X., McPartlin A. E., Brown L., Chen Y. H., Barker H. M., Cohen P. T. A novel human protein serine/threonine phosphatase, which possesses four tetratricopeptide repeat motifs and localizes to the nucleus EMBO J 1994 13, N 18 P. 4278– 4290.
[3] Blatch G. L., Lassle M. The tetratricopeptide repeat: a structural motif mediating protein-protein interactions Bioessays 1999 21, N 11 P. 932–939.
[4] Chen M. S., Silverstein A. M., Pratt W. B., Chinkers M. The tetratricopeptide repeat domain of protein phosphatase 5 mediates binding to glucocorticoid receptor heterocomplexes and acts as a dominant negative mutant J. Biol. Chem 1996 271, N 50 P. 32315–32320.
[5] Morita K., Saitoh M., Tobiume K., Matsuura H., Enomoto S., Nishitoh H., Ichijo H. Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress EMBO J 2001 20, N 21 P. 6028–6036.
[6] von Kriegsheim A., Pitt A., Grindlay G. J., Kolch W., Dhillon A. S. Regulation of the Raf-MEK-ERK pathway by protein phosphatase 5 Nat.Cell Biol 2006 8, N 9 P. 1011–1016.
[7] Chinkers M. Protein phosphatase 5 in signal transduction Trends Endocrinol. Metab 2001 12, N 1 P. 28–32.
[8] Yong W., Bao S., Chen H., Li D., Sanchez E. R., Shou W. Mice lacking protein phosphatase 5 are defective in ataxia telangiectasia mutated (ATM)-mediated cell cycle arrest J. Biol. Chem 2007 282, N 20 P. 14690–14694.
[9] Chen M. X., Cohen P. T. Activation of protein phosphatase 5 by limited proteolysis or the binding of polyunsaturated fatty acids to the TPR domain FEBS Lett 1997 400, N 1 P. 136– 140.
[10] Ghobrial I. M., McCormick D. J., Kaufmann S. H., Leontovich A. A., Loegering D. A., Dai N. T., Krajnik K. L., Stenson M. J., Melhem M. F., Novak A. J., Ansell S. M., Witzig T. E. Proteomic analysis of mantle-cell lymphoma by protein microarray Blood 2005 105, N 9 P. 3722–3730.
[11] Shirato H., Shima H., Nakagama H., Fukuda H., Watanabe Y., Ogawa K., Matsuda Y., Kikuchi K. Expression in hepatomas and chromosomal localization of rat protein phosphatase 5 gene Int. J. Oncol 2000 17, N 5 P. 909–912.
[12] Golden T., Aragon I. V., Rutland B., Tucker J. A., Shevde L. A., Samant R. S., Zhou G., Amable L., Skarra D., Honkanen R. E. Elevated levels of Ser/Thr protein phosphatase 5 (PP5) in human breast cancer Biochim. Biophys. Acta 2008 1782, N 4 P. 259–270.
[13] Golden T., Aragon I. V., Zhou G., Cooper S. R., Dean N. M., Honkanen R. E. Constitutive over expression of serine/threonine protein phosphatase 5 (PP5) augments estrogen-dependent tumor growth in mice Cancer Lett 2004 215, N 1 P. 95–100.
[14] Golden T., Swingle M., Honkanen R. E. The role of serine/threonine protein phosphatase type 5 (PP5) in the regulation of stressinduced signaling networks and cancer Cancer Metastasis Rev 2008 27, N 2–P. 169–178.
[15] Fukuda H., Tsuchiya N., Hara-Fujita K., Takagi S., Nagao M., Nakagama H. Induction of abnormal nuclear shapes in two distinct modes by overexpression of serine/threonine protein phosphatase 5 in Hela cells J. Cell Biochem 2007 101, N 2 P. 321–330.
[16] Vaughan C. K., Mollapour M., Smith J. R., Truman A., Hu B., Good V. M., Panaretou B., Neckers L., Clarke P. A., Workman P., Piper P. W., Prodromou C., Pearl L. H. Hsp90-dependent activation of protein kinases is regulated by chaperone-targeted dephosphorylation of Cdc37 Mol. Cell 2008 31, N 6 P. 886–895.
[17] Shao J., Hartson S. D., Matts R. L. Evidence that protein phosphatase 5 functions to negatively modulate the maturation of the Hsp90-dependent heme-regulated eIF2alpha kinase Biochemistry 2002 41, N 21 P. 6770–6779.
[18] Skarra D. V., Goudreault M., Choi H., Mullin M., Nesvizhskii A. I., Gingras A. C., Honkanen R. E. Label-free quantitative proteomics and SAINT analysis enable interactome mapping for the human Ser/Thr protein phosphatase 5 Proteomics 2011 11, N 8 P. 1508–1516.
[19] Mkaddem S. B., Werts C., Goujon J. M., Bens M., Pedruzzi E., Ogier-Denis E., Vandewalle A. Heat shock protein gp96 interacts with protein phosphatase 5 and controls toll-like receptor 2 (TLR2)-mediated activation of extracellular signal-regulated kinase (ERK) 1/2 in post-hypoxic kidney cells J. Biol. Chem 2009 284, N 18 P. 12541–12549.
[20] Zhou G., Golden T., Aragon I. V., Honkanen R. E. Ser/Thr protein phosphatase 5 inactivates hypoxia-induced activation of an apoptosis signal-regulating kinase 1/MKK-4/JNK signaling cascade J. Biol. Chem 2004 279, N 45 P. 46595–46605.
[21] Huang S., Shu L., Easton J., Harwood F. C., Germain G. S., Ichijo H., Houghton P. J. Inhibition of mammalian target of rapamycin activates apoptosis signal-regulating kinase 1 signaling by suppressing protein phosphatase 5 activity J. Biol. Chem 2004 279, N 35 P. 36490–36496.
[22] Zhang J., Bao S., Furumai R., Kucera K. S., Ali A., Dean N. M., Wang X. F. Protein phosphatase 5 is required for ATR-mediated checkpoint activation Mol. Cell Biol 2005 25, N 22 P. 9910–9919.
[23] Ali A., Zhang J., Bao S., Liu I., Otterness D., Dean N. M., Abraham R. T., Wang X. F. Requirement of protein phosphatase 5 in DNA-damage-induced ATM activation Genes Dev 2004 18, N 3 P. 249–254.
[24] Wechsler T., Chen B. P., Harper R., Morotomi-Yano K., Huang B. C., Meek K., Cleaver J. E., Chen D. J., Wabl M. DNA-PKcs function regulated specifically by protein phosphatase 5 Proc. Natl Acad. Sci. USA 2004 101, N 5 P. 1247–1252.
[25] Morita K., Saitoh M., Tobiume K., Matsuura H., Enomoto S., Nishitoh H., Ichijo H. Negative feedback regulation of ASK1 by protein phosphatase 5 (PP5) in response to oxidative stress EMBO J 2001 20, N 21 P. 6028–6036.
[26] Kutuzov M. A., Andreeva A. V., Voyno-Yasenetskaya T. A. Regulation of apoptosis signal-regulating kinase 1 (ASK1) by polyamine levels via protein phosphatase 5 J. Biol. Chem 2005 280, N 27 P. 25388–25395.
[27] Bruce D. L., Macartney T., Yong W., Shou W., Sapkota G. P. Protein phosphatase 5 modulates SMAD3 function in the transforming growth factor-b pathway Cell Signal 2012 24, N 11 P. 1999–2006.
[28] Malanchuk O. M., Pozur V., Panasyuk G. G., Nemazanyy I. O., Filonenko V. V., Gout I. T., Palchevskyy S. S. Identification of novel binding partners for tuberous sclerosis complex 2 (TSC2) by yeast two-hybrid approach Exp. Oncol 2005 27, N 3 P. 186–190.
[29] Malanchuk O. M., Palchevskyy S. S., Pozur V. K., Gout I. T., Filonenko V. V. Interaction of serine/threonine protein phosphatase 5 with the protein products of tumour suppressor gene Tsc2 Biopolym. Cell 2007 23, N 4 P. 318–323.
[30] Malanchuk O. M., Palchevskyy S. S., Filonenko V. V. Dephosphorylation of tuberous sclerosis complex 2 by serine/threonine protein phosphatase 5 Biopolym. Cell 2008 24, N 2 P. 176–179.
[31] Gao X., Pan D. TSC1 and TSC2 tumor suppressors antagonize insulin signaling in cell growth Genes Dev 2001 15, N 11 P. 1383–1392.
[32] Kwiatkowski D. J. Tuberous sclerosis: from tubers to mTOR Ann. Hum. Genet 2003 67, pt 1 P. 87–96.
[33] Harlow E., Lane D. Using antibodies: A laboratory manual New York: Cold Spring Harbor Lab. press, 1998 495 p.
[34] Hurrell J. G. R. Monoclonal hybridoma antibodies: Techniques and application Boca Raton: CRC press, 1982 231 p.