DOI QR코드

DOI QR Code

Phosphorylation on the PPP2R5D B regulatory subunit modulates the biochemical properties of protein phosphatase 2A

  • Yu, Un-Young (Department of Biochemistry, Ewha Womans University School of Medicine) ;
  • Ahn, Jung-Hyuck (Department of Biochemistry, Ewha Womans University School of Medicine)
  • Published : 2010.04.30

Abstract

To characterize the biochemical properties of the PP2A regulatory B subunit, PPP2R5D, we analyzed its phosphorylation sites, stoichiometry and effect on holoenzyme activity. PPP2R5D was phosphorylated on Ser-53, Ser-68, Ser-81, and Ser-566 by protein kinase A, and mutations at all four of these sites abolished any significant phosphorylation in vitro. In HEK293 cells, however, the Ser-566 was the major phosphorylation site after PKA activation by forskolin, with marginal phosphorylation on Ser-81. Inhibitory tyrosine phosphorylation on Tyr-307 of the PP2A catalytic C subunit was decreased after forskolin treatment. Kinetic analysis showed that overall PP2A activity was increased with phosphorylation by PPP2R5D phosphorylation. The apparent Km was reduced from $11.25\;{\mu}M$ to $1.175\;{\mu}M$ with PPP2R5D phosphorylation, resulting in an increase in catalytic activity. These data suggest that PKA-mediated activation of PP2A is enabled by PPP2R5D phosphorylation, which modulates the affinity of the PP2A holoenzyme to its physiological substrates.

Keywords

References

  1. Virshup, D. M. (2000) Protein phosphatase 2A: a panoply of enzymes. Curr. Opin. Cell. Biol. 12, 180-185 https://doi.org/10.1016/S0955-0674(99)00074-5
  2. Janssens, V. and Goris, J. (2001) Protein phosphatase 2A:a highly regulated family of serine/threonine phosphatases implicated in cell growth and signalling. Biochem. J. 353, 417-439 https://doi.org/10.1042/0264-6021:3530417
  3. Janssens, V., Longin, S. and Goris, J. (2008) PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail). Trends. Biochem. Sci. 33, 113-121 https://doi.org/10.1016/j.tibs.2007.12.004
  4. Margolis, S. S., Perry, J. A., Forester, C. M., Nutt, L. K., Guo, Y., Jardim, M. J., Thomenius, M. J., Freel, C. D., Darbandi, R., Ahn, J. H., Arroyo J. D., Wang X. F., Shenolikar, S., Nairn, A. C., Dunphy, W. G., Hahn, W. C., Virshup, D. M. and Kornbluth, S. (2006) Role for the PP2A/B56delta phosphatase in regulating 14-3-3 release from Cdc25 to control mitosis. Cell 127, 759-773 https://doi.org/10.1016/j.cell.2006.10.035
  5. Stipanovich, A., Valjent, E., Matamales, M., Nishi, A., Ahn, J. H., Maroteaux, M., Bertran-Gonzalez, J., Brami-Cherrier, K., Enslen, H., Corbille, A. G., Filhol O., Nairn A. C., Greengard, P., Heve, D. and Girault, J. A. (2008) A phosphatase cascade by which rewarding stimuli control nucleosomal response. Nature 453, 879-884 https://doi.org/10.1038/nature06994
  6. Ahn, J. H., McAvoy, T., Rakhilin, S. V., Nishi, A., Greengard, P. and Nairn, A. C. (2007) Protein kinase A activates protein phosphatase 2A by phosphorylation of the B56delta subunit. Proc. Natl. Acad. Sci. U.S.A. 104, 2979- 2984 https://doi.org/10.1073/pnas.0611532104
  7. Ahn, J. H., Sung, J. Y., McAvoy, T., Nishi, A., Janssens, V., Goris, J., Greengard, P. and Nairn, A. C. (2007) The B''/PR72 subunit mediates $Ca^{2+}$-dependent dephosphorylation of DARPP-32 by protein phosphatase 2A. Proc. Natl. Acad. Sci. U.S.A. 104, 9876-9881 https://doi.org/10.1073/pnas.0703589104
  8. Liu, R., Zhou, X. W., Tanila, H., Bjorkdahl, C., Wang, J. Z., Guan, Z. Z., Cao, Y., Gustafsson, J. A., Winblad, B. and Pei, J. J. (2008) Phosphorylated PP2A (tyrosine 307) is associated with Alzheimer neurofibrillary pathology. J. Cell. Mol. Med. 12, 241-257 https://doi.org/10.1111/j.1582-4934.2008.00249.x
  9. Usui, H., Inoue, R., Tanabe, O., Nishito, Y., Shimizu, M., Hayashi, H., Kagamiyama, H. and Takeda, M. (1998) Activation of protein phosphatase 2A by cAMP-dependent protein kinase-catalyzed phosphorylation of the 74-kDa B'' (delta) regulatory subunit in vitro and identification of the phosphorylation sites. FEBS Lett. 430, 312-316 https://doi.org/10.1016/S0014-5793(98)00684-X
  10. Sontag, E., Nunbhakdi-Craig, V., Sontag, J. M., Diaz-Arrastia, R., Ogris, E., Dayal, S., Lentz, S. R., Arning, E. and Bottiglieri, T. (2007) Protein phosphatase 2A methyltransferase links homocysteine metabolism with tau and amyloid precursor protein regulation. J. Neurosci. 27, 2751-2759 https://doi.org/10.1523/JNEUROSCI.3316-06.2007
  11. Vafai, S. B. and Stock, J. B. (2002) Protein phosphatase 2A methylation: a link between elevated plasma homocysteine and Alzheimer's disease. FEBS Lett. 518, 1-4 https://doi.org/10.1016/S0014-5793(02)02702-3
  12. Batut, J., Schmierer, B., Cao, J., Raftery, L. A., Hill, C. S. and Howell, M. (2008) Two highly related regulatory subunits of PP2A exert opposite effects on TGF-beta/Activin/Nodal signalling. Development 135, 2927-2937 https://doi.org/10.1242/dev.020842
  13. Fukui, R., Svenningsson, P., Matsuishi, T., Higashi, H., Nairn, A. C., Greengard, P. and Nishi, A. (2003) Effect of methylphenidate on dopamine/DARPP signalling in adult, but not young, mice. J. Neurochem. 87, 1391-1401 https://doi.org/10.1046/j.1471-4159.2003.02101.x
  14. Nishi, A., Bibb, J. A., Snyder, G. L., Higashi, H., Nairn, A. C. and Greengard, P. (2000) Amplification of dopaminergic signaling by a positive feedback loop. Proc. Natl. Acad. Sci. U.S.A. 97, 12840-12845 https://doi.org/10.1073/pnas.220410397
  15. Oh, M. K., Park, S. J., Kim N. H. and Kim, I. S. (2006) Protein kinase C-delta stimulates haptoglobin secretion. J. BMB 40, 130-134
  16. Bibb, J. A., Chen, J., Taylor, J. R., Svenningsson, P., Nishi, A., Snyder, G. L., Yan, Z., Sagawa, Z. K., Ouimet, C. C., Nairn, A. C., Nestler, E. J. and Greengard, P. (2001) Effects of chronic exposure to cocaine are regulated by the neuronal protein Cdk5. Nature 410, 376-380 https://doi.org/10.1038/35066591
  17. Azid, A. K. A., Sawa, Y., Ishikawa, T. and Shibata, H. (2006) Purification and characterization of protein phosphatase 2A from petals of tulip Tulipa gesnerina. J. BMB 39, 671-676

Cited by

  1. Proteomic Analysis of the Peri-Infarct Area after Human Umbilical Cord Mesenchymal Stem Cell Transplantation in Experimental Stroke vol.7, pp.5, 2016, https://doi.org/10.14336/AD.2016.0121
  2. Compact Modeling of Allosteric Multisite Proteins: Application to a Cell Size Checkpoint vol.10, pp.2, 2014, https://doi.org/10.1371/journal.pcbi.1003443
  3. Regulatory B Subunits of Protein Phosphatase 2A Are Involved in Site-specific Regulation of Tau Protein Phosphorylation vol.18, pp.2, 2014, https://doi.org/10.4196/kjpp.2014.18.2.155
  4. B56δ-related protein phosphatase 2A dysfunction identified in patients with intellectual disability vol.125, pp.8, 2015, https://doi.org/10.1172/JCI79860
  5. MicroRNA-9 regulates steroid-resistant airway hyperresponsiveness by reducing protein phosphatase 2A activity vol.136, pp.2, 2015, https://doi.org/10.1016/j.jaci.2014.11.044
  6. Identification of transcriptional and phosphatase regulators as interaction partners of human ADA3, a component of histone acetyltransferase complexes vol.450, pp.2, 2013, https://doi.org/10.1042/BJ20120452
  7. Determinants for Substrate Specificity of Protein Phosphatase 2A vol.2011, 2011, https://doi.org/10.4061/2011/398751
  8. Identification and association analysis of several hundred single nucleotide polymorphisms within candidate genes for back fat thickness in Italian Large White pigs using a selective genotyping approach1 vol.90, pp.8, 2012, https://doi.org/10.2527/jas.2011-4797
  9. G protein–dependent signaling triggers a β-arrestin–scaffolded p70S6K/ rpS6 module that controls 5′TOP mRNA translation vol.32, pp.3, 2018, https://doi.org/10.1096/fj.201700763R