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J Virol. Jan 1990; 64(1): 61–68.
PMCID: PMC249044

Regulation of pp60c-src and its interaction with polyomavirus middle T antigen in insect cells.

Abstract

High yields of soluble, biologically active pp60c-src and middle t antigen (MTAg) of polyomavirus were produced in insect cells, using a baculovirus expression system. In mammalian cells, pp60c-src undergoes a regulatory phosphorylation on Tyr-527 in vivo and is autophosphorylated on Tyr-416 in vitro. In insect cells, pp60c-src was phosphorylated primarily on Tyr-416, although Tyr-527 was detectable at a low level. A kinase-negative mutant of pp60c-src was not phosphorylated on either Tyr-527 or Tyr-416 in insect cells and thus is an excellent biochemical reagent to search for the regulatory kinase that usually phosphorylates Tyr-527 in mammalian cells. MTAg synthesized in insect cells was not phosphorylated on tyrosine residues in vivo or in vitro, suggesting that it did not associate with any endogenous tyrosine kinases. However, MTAg isolated from cells coinfected with viruses encoding both MTAg and pp60c-src was phosphorylated on tyrosine residues both in vivo and in vitro.

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  • Bolen JB, DeSeau V, O'Shaughnessy J, Amini S. Analysis of middle tumor antigen and pp60c-src interactions in polyomavirus-transformed rat cells. J Virol. 1987 Oct;61(10):3299–3305. [PMC free article] [PubMed]
  • Bolen JB, Thiele CJ, Israel MA, Yonemoto W, Lipsich LA, Brugge JS. Enhancement of cellular src gene product associated tyrosyl kinase activity following polyoma virus infection and transformation. Cell. 1984 Oct;38(3):767–777. [PubMed]
  • Buss JE, Sefton BM. Myristic acid, a rare fatty acid, is the lipid attached to the transforming protein of Rous sarcoma virus and its cellular homolog. J Virol. 1985 Jan;53(1):7–12. [PMC free article] [PubMed]
  • Carbonell LF, Klowden MJ, Miller LK. Baculovirus-mediated expression of bacterial genes in dipteran and mammalian cells. J Virol. 1985 Oct;56(1):153–160. [PMC free article] [PubMed]
  • Cartwright CA, Eckhart W, Simon S, Kaplan PL. Cell transformation by pp60c-src mutated in the carboxy-terminal regulatory domain. Cell. 1987 Apr 10;49(1):83–91. [PubMed]
  • Cartwright CA, Kaplan PL, Cooper JA, Hunter T, Eckhart W. Altered sites of tyrosine phosphorylation in pp60c-src associated with polyomavirus middle tumor antigen. Mol Cell Biol. 1986 May;6(5):1562–1570. [PMC free article] [PubMed]
  • Cheng SH, Harvey R, Espino PC, Semba K, Yamamoto T, Toyoshima K, Smith AE. Peptide antibodies to the human c-fyn gene product demonstrate pp59c-fyn is capable of complex formation with the middle-T antigen of polyomavirus. EMBO J. 1988 Dec 1;7(12):3845–3855. [PMC free article] [PubMed]
  • Cheng SH, Markland W, Markham AF, Smith AE. Mutations around the NG59 lesion indicate an active association of polyoma virus middle-T antigen with pp60c-src is required for cell transformation. EMBO J. 1986 Feb;5(2):325–334. [PMC free article] [PubMed]
  • Cheng SH, Piwnica-Worms H, Harvey RW, Roberts TM, Smith AE. The carboxy terminus of pp60c-src is a regulatory domain and is involved in complex formation with the middle-T antigen of polyomavirus. Mol Cell Biol. 1988 Apr;8(4):1736–1747. [PMC free article] [PubMed]
  • Cherington V, Morgan B, Spiegelman BM, Roberts TM. Recombinant retroviruses that transduce individual polyoma tumor antigens: effects on growth and differentiation. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4307–4311. [PMC free article] [PubMed]
  • Collett MS, Erikson E, Erikson RL. Structural analysis of the avian sarcoma virus transforming protein: sites of phosphorylation. J Virol. 1979 Feb;29(2):770–781. [PMC free article] [PubMed]
  • Collett MS, Purchio AF, Erikson RL. Avian sarcoma virus-transforming protein, pp60src shows protein kinase activity specific for tyrosine. Nature. 1980 May 15;285(5761):167–169. [PubMed]
  • Cooper JA, Gould KL, Cartwright CA, Hunter T. Tyr527 is phosphorylated in pp60c-src: implications for regulation. Science. 1986 Mar 21;231(4744):1431–1434. [PubMed]
  • Cooper JA, King CS. Dephosphorylation or antibody binding to the carboxy terminus stimulates pp60c-src. Mol Cell Biol. 1986 Dec;6(12):4467–4477. [PMC free article] [PubMed]
  • Cooper JA, MacAuley A. Potential positive and negative autoregulation of p60c-src by intermolecular autophosphorylation. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4232–4236. [PMC free article] [PubMed]
  • Cooper JA, Runge K. Avian pp60c-src is more active when expressed in yeast than in vertebrate fibroblasts. Oncogene Res. 1987 Sep-Oct;1(4):297–310. [PubMed]
  • Courtneidge SA. Activation of the pp60c-src kinase by middle T antigen binding or by dephosphorylation. EMBO J. 1985 Jun;4(6):1471–1477. [PMC free article] [PubMed]
  • Courtneidge SA, Heber A. An 81 kd protein complexed with middle T antigen and pp60c-src: a possible phosphatidylinositol kinase. Cell. 1987 Sep 25;50(7):1031–1037. [PubMed]
  • Courtneidge SA, Smith AE. Polyoma virus transforming protein associates with the product of the c-src cellular gene. Nature. 1983 Jun 2;303(5916):435–439. [PubMed]
  • Courtneidge SA, Smith AE. The complex of polyoma virus middle-T antigen and pp60c-src. EMBO J. 1984 Mar;3(3):585–591. [PMC free article] [PubMed]
  • Coussens PM, Cooper JA, Hunter T, Shalloway D. Restriction of the in vitro and in vivo tyrosine protein kinase activities of pp60c-src relative to pp60v-src. Mol Cell Biol. 1985 Oct;5(10):2753–2763. [PMC free article] [PubMed]
  • Cross FR, Hanafusa H. Local mutagenesis of Rous sarcoma virus: the major sites of tyrosine and serine phosphorylation of pp60src are dispensable for transformation. Cell. 1983 Sep;34(2):597–607. [PubMed]
  • Hunter T, Sefton BM. Transforming gene product of Rous sarcoma virus phosphorylates tyrosine. Proc Natl Acad Sci U S A. 1980 Mar;77(3):1311–1315. [PMC free article] [PubMed]
  • Iba H, Cross FR, Garber EA, Hanafusa H. Low level of cellular protein phosphorylation by nontransforming overproduced p60c-src. Mol Cell Biol. 1985 May;5(5):1058–1066. [PMC free article] [PubMed]
  • Jove R, Kornbluth S, Hanafusa H. Enzymatically inactive p60c-src mutant with altered ATP-binding site is fully phosphorylated in its carboxy-terminal regulatory region. Cell. 1987 Sep 11;50(6):937–943. [PubMed]
  • Kaplan DR, Bockus B, Roberts TM, Bolen J, Israel M, Schaffhausen BS. Large-scale production of polyoma middle T antigen by using genetically engineered tumors. Mol Cell Biol. 1985 Jul;5(7):1795–1799. [PMC free article] [PubMed]
  • Kaplan DR, Whitman M, Schaffhausen B, Pallas DC, White M, Cantley L, Roberts TM. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987 Sep 25;50(7):1021–1029. [PubMed]
  • Kmiecik TE, Shalloway D. Activation and suppression of pp60c-src transforming ability by mutation of its primary sites of tyrosine phosphorylation. Cell. 1987 Apr 10;49(1):65–73. [PubMed]
  • Kornbluth S, Jove R, Hanafusa H. Characterization of avian and viral p60src proteins expressed in yeast. Proc Natl Acad Sci U S A. 1987 Jul;84(13):4455–4459. [PMC free article] [PubMed]
  • Kornbluth S, Sudol M, Hanafusa H. Association of the polyomavirus middle-T antigen with c-yes protein. Nature. 1987 Jan 8;325(7000):171–173. [PubMed]
  • Kypta RM, Hemming A, Courtneidge SA. Identification and characterization of p59fyn (a src-like protein tyrosine kinase) in normal and polyoma virus transformed cells. EMBO J. 1988 Dec 1;7(12):3837–3844. [PMC free article] [PubMed]
  • Levinson AD, Oppermann H, Varmus HE, Bishop JM. The purified product of the transforming gene of avian sarcoma virus phosphorylates tyrosine. J Biol Chem. 1980 Dec 25;255(24):11973–11980. [PubMed]
  • Pallas DC, Cherington V, Morgan W, DeAnda J, Kaplan D, Schaffhausen B, Roberts TM. Cellular proteins that associate with the middle and small T antigens of polyomavirus. J Virol. 1988 Nov;62(11):3934–3940. [PMC free article] [PubMed]
  • Pallas DC, Schley C, Mahoney M, Harlow E, Schaffhausen BS, Roberts TM. Polyomavirus small t antigen: overproduction in bacteria, purification, and utilization for monoclonal and polyclonal antibody production. J Virol. 1986 Dec;60(3):1075–1084. [PMC free article] [PubMed]
  • Pallas D, Solomon F. Cytoplasmic microtubule-associated proteins: phosphorylation at novel sites is correlated with their incorporation into assembled microtubules. Cell. 1982 Sep;30(2):407–414. [PubMed]
  • Parsons SJ, McCarley DJ, Ely CM, Benjamin DC, Parsons JT. Monoclonal antibodies to Rous sarcoma virus pp60src react with enzymatically active cellular pp60src of avian and mammalian origin. J Virol. 1984 Aug;51(2):272–282. [PMC free article] [PubMed]
  • Patschinsky T, Hunter T, Esch FS, Cooper JA, Sefton BM. Analysis of the sequence of amino acids surrounding sites of tyrosine phosphorylation. Proc Natl Acad Sci U S A. 1982 Feb;79(4):973–977. [PMC free article] [PubMed]
  • Patschinsky T, Hunter T, Sefton BM. Phosphorylation of the transforming protein of Rous sarcoma virus: direct demonstration of phosphorylation of serine 17 and identification of an additional site of tyrosine phosphorylation in p60v-src of Prague Rous sarcoma virus. J Virol. 1986 Jul;59(1):73–81. [PMC free article] [PubMed]
  • Piwnica-Worms H, Kaplan DR, Whitman M, Roberts TM. Retrovirus shuttle vector for study of kinase activities of pp60c-src synthesized in vitro and overproduced in vivo. Mol Cell Biol. 1986 Jun;6(6):2033–2040. [PMC free article] [PubMed]
  • Piwnica-Worms H, Saunders KB, Roberts TM, Smith AE, Cheng SH. Tyrosine phosphorylation regulates the biochemical and biological properties of pp60c-src. Cell. 1987 Apr 10;49(1):75–82. [PubMed]
  • Raptis L, Lamfrom H, Benjamin TL. Regulation of cellular phenotype and expression of polyomavirus middle T antigen in rat fibroblasts. Mol Cell Biol. 1985 Sep;5(9):2476–2486. [PMC free article] [PubMed]
  • Schaffhausen B, Benjamin TL. Comparison of phosphorylation of two polyoma virus middle T antigens in vivo and in vitro. J Virol. 1981 Oct;40(1):184–196. [PMC free article] [PubMed]
  • Schaffhausen BS, Bockus BJ, Berkner KL, Kaplan D, Roberts TM. Characterization of middle T antigen expressed by using an adenovirus expression system. J Virol. 1987 Apr;61(4):1221–1225. [PMC free article] [PubMed]
  • Schultz AM, Henderson LE, Oroszlan S, Garber EA, Hanafusa H. Amino terminal myristylation of the protein kinase p60src, a retroviral transforming protein. Science. 1985 Jan 25;227(4685):427–429. [PubMed]
  • Simon MA, Drees B, Kornberg T, Bishop JM. The nucleotide sequence and the tissue-specific expression of Drosophila c-src. Cell. 1985 Oct;42(3):831–840. [PubMed]
  • Smart JE, Oppermann H, Czernilofsky AP, Purchio AF, Erikson RL, Bishop JM. Characterization of sites for tyrosine phosphorylation in the transforming protein of Rous sarcoma virus (pp60v-src) and its normal cellular homologue (pp60c-src). Proc Natl Acad Sci U S A. 1981 Oct;78(10):6013–6017. [PMC free article] [PubMed]
  • Snyder MA, Bishop JM, McGrath JP, Levinson AD. A mutation at the ATP-binding site of pp60v-src abolishes kinase activity, transformation, and tumorigenicity. Mol Cell Biol. 1985 Jul;5(7):1772–1779. [PMC free article] [PubMed]
  • Yonemoto W, Jarvis-Morar M, Brugge JS, Bolen JB, Israel MA. Tyrosine phosphorylation within the amino-terminal domain of pp60c-src molecules associated with polyoma virus middle-sized tumor antigen. Proc Natl Acad Sci U S A. 1985 Jul;82(14):4568–4572. [PMC free article] [PubMed]

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