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. 1986 Jan;83(2):207–211. doi: 10.1073/pnas.83.2.207

Phosphorylation and inactivation of protein phosphatase 1 by pp60v-src.

J W Johansen, T S Ingebritsen
PMCID: PMC322826  PMID: 3001727

Abstract

Protein phosphatase 1, one of four major protein phosphatases involved in cellular regulation, was phosphorylated in vitro by pp60v-src, the transforming gene product of Rous sarcoma virus. Phosphorylation was accompanied by a loss of protein phosphatase activity. The inactivation of protein phosphatase 1 was time-dependent and the extent of inactivation correlated closely with the stoichiometry of phosphorylation. Under optimal conditions, 0.34 +/- 0.01 mol of phosphate were incorporated per mol of protein phosphatase and the activity of the enzyme was decreased by 39 +/- 2%. The inactivation required the presence of both MgATP and pp60v-src. There was no loss of activity when adenosine 5'-[beta gamma-imido]triphosphate was used in place of ATP. Phosphorylation of protein phosphatase 1 occurred exclusively on tyrosine residues and was blocked by specific antibodies to pp60v-src. During preincubation of pp60v-src at 41 degrees C, its protein kinase activity towards casein was lost rapidly. The ability of pp60v-src to phosphorylate and inactivate protein phosphatase 1 declined in parallel with the loss of casein kinase activity. Limited chymotryptic digestion of 32P-labeled protein phosphatase 1 (Mr 37,000) resulted in its quantitative conversion to a Mr 33,000 species. Conversion to this species was accompanied by the loss of 32P-labeling and by reactivation of the protein phosphatase. When various concentrations of chymotrypsin were used in the digestion, there was a close correlation between conversion to the Mr 33,000 species and the restoration of protein phosphatase activity. pp60v-src was unable to phosphorylate or inactivate a partially proteolyzed species of protein phosphatase 1 (Mr 33,000/34,000).

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Selected References

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  1. Berridge M. J. Inositol trisphosphate and diacylglycerol as second messengers. Biochem J. 1984 Jun 1;220(2):345–360. doi: 10.1042/bj2200345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  3. Burchell A., Foulkes J. G., Cohen P. T., Condon G. D., Cohen P. Evidence for the involvement of protein phosphatase-1 in the regulation of metabolic processes other than glycogen metabolism. FEBS Lett. 1978 Aug 1;92(1):68–72. doi: 10.1016/0014-5793(78)80723-6. [DOI] [PubMed] [Google Scholar]
  4. Cohen P. The role of protein phosphorylation in neural and hormonal control of cellular activity. Nature. 1982 Apr 15;296(5858):613–620. doi: 10.1038/296613a0. [DOI] [PubMed] [Google Scholar]
  5. Cohen P. The subunit structure of rabbit-skeletal-muscle phosphorylase kinase, and the molecular basis of its activation reactions. Eur J Biochem. 1973 Apr 2;34(1):1–14. doi: 10.1111/j.1432-1033.1973.tb02721.x. [DOI] [PubMed] [Google Scholar]
  6. Cooper J. A., Hunter T. Changes in protein phosphorylation in Rous sarcoma virus-transformed chicken embryo cells. Mol Cell Biol. 1981 Feb;1(2):165–178. doi: 10.1128/mcb.1.2.165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cooper J. A., Hunter T. Regulation of cell growth and transformation by tyrosine-specific protein kinases: the search for important cellular substrate proteins. Curr Top Microbiol Immunol. 1983;107:125–161. doi: 10.1007/978-3-642-69075-4_4. [DOI] [PubMed] [Google Scholar]
  8. Decker S. Phosphorylation of ribosomal protein S6 in avian sarcoma virus-transformed chicken embryo fibroblasts. Proc Natl Acad Sci U S A. 1981 Jul;78(7):4112–4115. doi: 10.1073/pnas.78.7.4112. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Diringer H., Friis R. R. Changes in phosphatidylinositol metabolism correlated to growth state of normal and Rous sarcoma virus-transformed Japanese quail cells. Cancer Res. 1977 Sep;37(9):2979–2984. [PubMed] [Google Scholar]
  10. Erikson R. I., Collett M. S., Erikson E., Purchio A. F., Brugge J. S. Protein phosphorylation mediated by partially purified avian sarcoma virus transforming-gene product. Cold Spring Harb Symp Quant Biol. 1980;44(Pt 2):907–917. doi: 10.1101/sqb.1980.044.01.098. [DOI] [PubMed] [Google Scholar]
  11. Erikson R. L., Collett M. S., Erikson E., Purchio A. F. Evidence that the avian sarcoma virus transforming gene product is a cyclic AMP-independent protein kinase. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6260–6264. doi: 10.1073/pnas.76.12.6260. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. FISCHER E. H., KREBS E. G. The isolation and crystallization of rabbit skeletal muscle phosphorylase b. J Biol Chem. 1958 Mar;231(1):65–71. [PubMed] [Google Scholar]
  13. Foulkes J. G., Cohen P. The hormonal control of glycogen metabolism. Phosphorylation of protein phosphatase inhibitor-1 in vivo in response to adrenaline. Eur J Biochem. 1979 Jun;97(1):251–256. doi: 10.1111/j.1432-1033.1979.tb13109.x. [DOI] [PubMed] [Google Scholar]
  14. Guy P. S., Cohen P., Hardie D. G. Purification and physicochemical properties of ATP citrate (pro-3S) lyase from lactating rat mammary gland and studies of its reversible phosphorylation. Eur J Biochem. 1981 Feb;114(2):399–405. doi: 10.1111/j.1432-1033.1981.tb05160.x. [DOI] [PubMed] [Google Scholar]
  15. Hazra A. K., Chock S. P., Albers R. W. Protein determination with trinitrobenzene sulfonate: a method relatively independent of amino acid composition. Anal Biochem. 1984 Mar;137(2):437–443. doi: 10.1016/0003-2697(84)90110-6. [DOI] [PubMed] [Google Scholar]
  16. Hemmings B. A., Resink T. J., Cohen P. Reconstitution of a Mg-ATP-dependent protein phosphatase and its activation through a phosphorylation mechanism. FEBS Lett. 1982 Dec 27;150(2):319–324. doi: 10.1016/0014-5793(82)80760-6. [DOI] [PubMed] [Google Scholar]
  17. Hemmings H. C., Jr, Greengard P., Tung H. Y., Cohen P. DARPP-32, a dopamine-regulated neuronal phosphoprotein, is a potent inhibitor of protein phosphatase-1. Nature. 1984 Aug 9;310(5977):503–505. doi: 10.1038/310503a0. [DOI] [PubMed] [Google Scholar]
  18. Hemmings H. C., Jr, Nairn A. C., Aswad D. W., Greengard P. DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. II. Purification and characterization of the phosphoprotein from bovine caudate nucleus. J Neurosci. 1984 Jan;4(1):99–110. doi: 10.1523/JNEUROSCI.04-01-00099.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Hemmings H. C., Jr, Williams K. R., Konigsberg W. H., Greengard P. DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated neuronal phosphoprotein. I. Amino acid sequence around the phosphorylated threonine. J Biol Chem. 1984 Dec 10;259(23):14486–14490. [PubMed] [Google Scholar]
  20. Huang F. L., Glinsmann W. H. Separation and characterization of two phosphorylase phosphatase inhibitors from rabbit skeletal muscle. Eur J Biochem. 1976 Nov 15;70(2):419–426. doi: 10.1111/j.1432-1033.1976.tb11032.x. [DOI] [PubMed] [Google Scholar]
  21. Hunter T., Sefton B. M. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. doi: 10.1073/pnas.77.3.1311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Ingebritsen T. S., Cohen P. Protein phosphatases: properties and role in cellular regulation. Science. 1983 Jul 22;221(4608):331–338. doi: 10.1126/science.6306765. [DOI] [PubMed] [Google Scholar]
  23. Ingebritsen T. S., Stewart A. A., Cohen P. The protein phosphatases involved in cellular regulation. 6. Measurement of type-1 and type-2 protein phosphatases in extracts of mammalian tissues; an assessment of their physiological roles. Eur J Biochem. 1983 May 2;132(2):297–307. doi: 10.1111/j.1432-1033.1983.tb07362.x. [DOI] [PubMed] [Google Scholar]
  24. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  25. Nimmo G. A., Cohen P. The regulation of glycogen metabolism. Phosphorylation of inhibitor-1 from rabbit skeletal muscle, and its interaction with protein phosphatases-III and -II. Eur J Biochem. 1978 Jun 15;87(2):353–365. doi: 10.1111/j.1432-1033.1978.tb12384.x. [DOI] [PubMed] [Google Scholar]
  26. Nimmo G. A., Cohen P. The regulation of glycogen metabolism. Purification and characterisation of protein phosphatase inhibitor-1 from rabbit skeletal muscle. Eur J Biochem. 1978 Jun 15;87(2):341–351. doi: 10.1111/j.1432-1033.1978.tb12383.x. [DOI] [PubMed] [Google Scholar]
  27. Nishizuka Y. The role of protein kinase C in cell surface signal transduction and tumour promotion. Nature. 1984 Apr 19;308(5961):693–698. doi: 10.1038/308693a0. [DOI] [PubMed] [Google Scholar]
  28. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  29. Purchio A. F., Erikson E., Brugge J. S., Erikson R. L. Identification of a polypeptide encoded by the avian sarcoma virus src gene. Proc Natl Acad Sci U S A. 1978 Mar;75(3):1567–1571. doi: 10.1073/pnas.75.3.1567. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Resink T. J., Hemmings B. A., Tung H. Y., Cohen P. Characterisation of a reconstituted Mg-ATP-dependent protein phosphatase. Eur J Biochem. 1983 Jun 15;133(2):455–461. doi: 10.1111/j.1432-1033.1983.tb07485.x. [DOI] [PubMed] [Google Scholar]
  31. Sefton B. M., Hunter T., Beemon K., Eckhart W. Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus. Cell. 1980 Jul;20(3):807–816. doi: 10.1016/0092-8674(80)90327-x. [DOI] [PubMed] [Google Scholar]
  32. Sefton B. M., Hunter T. Tyrosine protein kinases. Adv Cyclic Nucleotide Protein Phosphorylation Res. 1984;18:195–226. [PubMed] [Google Scholar]
  33. Shenolikar S., Ingebritsen T. S. Protein (serine and threonine) phosphate phosphatases. Methods Enzymol. 1984;107:102–129. doi: 10.1016/0076-6879(84)07007-5. [DOI] [PubMed] [Google Scholar]
  34. Sugimoto Y., Whitman M., Cantley L. C., Erikson R. L. Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2117–2121. doi: 10.1073/pnas.81.7.2117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Tung H. Y., Resink T. J., Hemmings B. A., Shenolikar S., Cohen P. The catalytic subunits of protein phosphatase-1 and protein phosphatase 2A are distinct gene products. Eur J Biochem. 1984 Feb 1;138(3):635–641. doi: 10.1111/j.1432-1033.1984.tb07962.x. [DOI] [PubMed] [Google Scholar]
  36. Usa M., Ishimura K., Fujita H., Sugano S., Okamoto M., Yamano T. Ultrastructural and immunohistochemical studies on the zona-reticularis cells of the adrenal cortex of normal and 3-methylcholanthrene-treated mice. Histochemistry. 1985;83(3):207–211. doi: 10.1007/BF00953985. [DOI] [PubMed] [Google Scholar]
  37. Villa-Moruzzi E., Ballou L. M., Fischer E. H. Phosphorylase phosphatase. Interconversion of active and inactive forms. J Biol Chem. 1984 May 10;259(9):5857–5863. [PubMed] [Google Scholar]
  38. Walaas S. I., Greengard P. DARPP-32, a dopamine- and adenosine 3':5'-monophosphate-regulated phosphoprotein enriched in dopamine-innervated brain regions. I. Regional and cellular distribution in the rat brain. J Neurosci. 1984 Jan;4(1):84–98. doi: 10.1523/JNEUROSCI.04-01-00084.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Walseth T. F., Johnson R. A. The enzymatic preparation of [alpha-(32)P]nucleoside triphosphates, cyclic [32P] AMP, and cyclic [32P] GMP. Biochim Biophys Acta. 1979 Mar 28;562(1):11–31. doi: 10.1016/0005-2787(79)90122-9. [DOI] [PubMed] [Google Scholar]

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