PMCCPMCCPMCC

Search tips
Search criteria 

Advanced

 
Logo of molcellbPermissionsJournals.ASM.orgJournalMCB ArticleJournal InfoAuthorsReviewers
 
Mol Cell Biol. 1993 November; 13(11): 7112–7121.
PMCID: PMC364772

The SH2 domain is required for stable phosphorylation of p56lck at tyrosine 505, the negative regulatory site.

Abstract

The catalytic function of Src-related tyrosine protein kinases is repressed by phosphorylation of a conserved carboxy-terminal tyrosine residue. Recent studies suggest that this inhibitory event is not the result of autophosphorylation but that it is mediated by another cytoplasmic tyrosine protein kinase, termed p50csk. In this report, we have evaluated the processes regulating the extent of phosphorylation of the inhibitory carboxy-terminal tyrosine residue of p56lck, a lymphocyte-specific member of the Src family. By analyzing kinase-defective variants of p56lck expressed in mouse NIH 3T3 cells, we have found that the noncatalytic Src homology 2 (SH2) domain, but not the SH3 sequence or the sites of Lck myristylation and autophosphorylation, is necessary for stable phosphorylation at the carboxy-terminal tyrosine 505. Further studies in which Lck and Csk were coexpressed in S. cerevisiae indicated that the absence of the SH2 domain did not affect the ability of Csk to phosphorylate p56lck at tyrosine 505. However, we observed that incubation of cells with the tyrosine phosphatase inhibitor pervanadate restored the tyrosine 505 phosphorylation of Lck polypeptides devoid of the SH2 motif. Additionally, the presence of the SH2 sequence protected tyrosine 505 from in vitro dephosphorylation by the hemopoietic tyrosine protein phosphatase CD45. Taken together, these findings raised the possibility that the SH2 motif contributes to the physiological suppression of the catalytic function of p56lck at least in part through its ability to stabilize phosphorylation at the inhibitory site.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (2.3M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Images in this article

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Abraham N, Miceli MC, Parnes JR, Veillette A. Enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Nature. 1991 Mar 7;350(6313):62–66. [PubMed]
  • Abraham N, Veillette A. Activation of p56lck through mutation of a regulatory carboxy-terminal tyrosine residue requires intact sites of autophosphorylation and myristylation. Mol Cell Biol. 1990 Oct;10(10):5197–5206. [PMC free article] [PubMed]
  • Albritton LM, Tseng L, Scadden D, Cunningham JM. A putative murine ecotropic retrovirus receptor gene encodes a multiple membrane-spanning protein and confers susceptibility to virus infection. Cell. 1989 May 19;57(4):659–666. [PubMed]
  • Amrein KE, Sefton BM. Mutation of a site of tyrosine phosphorylation in the lymphocyte-specific tyrosine protein kinase, p56lck, reveals its oncogenic potential in fibroblasts. Proc Natl Acad Sci U S A. 1988 Jun;85(12):4247–4251. [PubMed]
  • Bagrodia S, Chackalaparampil I, Kmiecik TE, Shalloway D. Altered tyrosine 527 phosphorylation and mitotic activation of p60c-src. Nature. 1991 Jan 10;349(6305):172–175. [PubMed]
  • Bergman M, Mustelin T, Oetken C, Partanen J, Flint NA, Amrein KE, Autero M, Burn P, Alitalo K. The human p50csk tyrosine kinase phosphorylates p56lck at Tyr-505 and down regulates its catalytic activity. EMBO J. 1992 Aug;11(8):2919–2924. [PubMed]
  • Cantley LC, Auger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. [PubMed]
  • Caron L, Abraham N, Pawson T, Veillette A. Structural requirements for enhancement of T-cell responsiveness by the lymphocyte-specific tyrosine protein kinase p56lck. Mol Cell Biol. 1992 Jun;12(6):2720–2729. [PMC free article] [PubMed]
  • Cartier M, Chang MW, Stanners CP. Use of the Escherichia coli gene for asparagine synthetase as a selective marker in a shuttle vector capable of dominant transfection and amplification in animal cells. Mol Cell Biol. 1987 May;7(5):1623–1628. [PMC free article] [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]
  • Felder S, Zhou M, Hu P, Ureña J, Ullrich A, Chaudhuri M, White M, Shoelson SE, Schlessinger J. SH2 domains exhibit high-affinity binding to tyrosine-phosphorylated peptides yet also exhibit rapid dissociation and exchange. Mol Cell Biol. 1993 Mar;13(3):1449–1455. [PMC free article] [PubMed]
  • Hurley TR, Hyman R, Sefton BM. Differential effects of expression of the CD45 tyrosine protein phosphatase on the tyrosine phosphorylation of the lck, fyn, and c-src tyrosine protein kinases. Mol Cell Biol. 1993 Mar;13(3):1651–1656. [PMC free article] [PubMed]
  • Ito H, Fukuda Y, Murata K, Kimura A. Transformation of intact yeast cells treated with alkali cations. J Bacteriol. 1983 Jan;153(1):163–168. [PMC free article] [PubMed]
  • Koch CA, Anderson D, Moran MF, Ellis C, Pawson T. SH2 and SH3 domains: elements that control interactions of cytoplasmic signaling proteins. Science. 1991 May 3;252(5006):668–674. [PubMed]
  • Liu X, Brodeur SR, Gish G, Songyang Z, Cantley LC, Laudano AP, Pawson T. Regulation of c-Src tyrosine kinase activity by the Src SH2 domain. Oncogene. 1993 May;8(5):1119–1126. [PubMed]
  • Marth JD, Cooper JA, King CS, Ziegler SF, Tinker DA, Overell RW, Krebs EG, Perlmutter RM. Neoplastic transformation induced by an activated lymphocyte-specific protein tyrosine kinase (pp56lck). Mol Cell Biol. 1988 Feb;8(2):540–550. [PMC free article] [PubMed]
  • Marth JD, Peet R, Krebs EG, Perlmutter RM. A lymphocyte-specific protein-tyrosine kinase gene is rearranged and overexpressed in the murine T cell lymphoma LSTRA. Cell. 1985 Dec;43(2 Pt 1):393–404. [PubMed]
  • Miller AD, Rosman GJ. Improved retroviral vectors for gene transfer and expression. Biotechniques. 1989 Oct;7(9):980–990. [PMC free article] [PubMed]
  • Mustelin T, Burn P. Regulation of src family tyrosine kinases in lymphocytes. Trends Biochem Sci. 1993 Jun;18(6):215–220. [PubMed]
  • Nada S, Okada M, MacAuley A, Cooper JA, Nakagawa H. Cloning of a complementary DNA for a protein-tyrosine kinase that specifically phosphorylates a negative regulatory site of p60c-src. Nature. 1991 May 2;351(6321):69–72. [PubMed]
  • Okada M, Nada S, Yamanashi Y, Yamamoto T, Nakagawa H. CSK: a protein-tyrosine kinase involved in regulation of src family kinases. J Biol Chem. 1991 Dec 25;266(36):24249–24252. [PubMed]
  • O'Shea JJ, McVicar DW, Bailey TL, Burns C, Smyth MJ. Activation of human peripheral blood T lymphocytes by pharmacological induction of protein-tyrosine phosphorylation. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10306–10310. [PubMed]
  • Ostergaard HL, Shackelford DA, Hurley TR, Johnson P, Hyman R, Sefton BM, Trowbridge IS. Expression of CD45 alters phosphorylation of the lck-encoded tyrosine protein kinase in murine lymphoma T-cell lines. Proc Natl Acad Sci U S A. 1989 Nov;86(22):8959–8963. [PubMed]
  • Pawson T, Gish GD. SH2 and SH3 domains: from structure to function. Cell. 1992 Oct 30;71(3):359–362. [PubMed]
  • Payne G, Shoelson SE, Gish GD, Pawson T, Walsh CT. Kinetics of p56lck and p60src Src homology 2 domain binding to tyrosine-phosphorylated peptides determined by a competition assay or surface plasmon resonance. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):4902–4906. [PubMed]
  • Reske-Kunz AB, Rüde E. Insulin-specific T cell hybridomas derived from (H-2b x H-2k)F1 mice preferably employ F1-unique restriction elements for antigen recognition. Eur J Immunol. 1985 Oct;15(10):1048–1054. [PubMed]
  • Rotin D, Margolis B, Mohammadi M, Daly RJ, Daum G, Li N, Fischer EH, Burgess WH, Ullrich A, Schlessinger J. SH2 domains prevent tyrosine dephosphorylation of the EGF receptor: identification of Tyr992 as the high-affinity binding site for SH2 domains of phospholipase C gamma. EMBO J. 1992 Feb;11(2):559–567. [PubMed]
  • Roussel RR, Brodeur SR, Shalloway D, Laudano AP. Selective binding of activated pp60c-src by an immobilized synthetic phosphopeptide modeled on the carboxyl terminus of pp60c-src. Proc Natl Acad Sci U S A. 1991 Dec 1;88(23):10696–10700. [PubMed]
  • Sabe H, Okada M, Nakagawa H, Hanafusa H. Activation of c-Src in cells bearing v-Crk and its suppression by Csk. Mol Cell Biol. 1992 Oct;12(10):4706–4713. [PMC free article] [PubMed]
  • Secrist JP, Burns LA, Karnitz L, Koretzky GA, Abraham RT. Stimulatory effects of the protein tyrosine phosphatase inhibitor, pervanadate, on T-cell activation events. J Biol Chem. 1993 Mar 15;268(8):5886–5893. [PubMed]
  • Seidel-Dugan C, Meyer BE, Thomas SM, Brugge JS. Effects of SH2 and SH3 deletions on the functional activities of wild-type and transforming variants of c-Src. Mol Cell Biol. 1992 Apr;12(4):1835–1845. [PMC free article] [PubMed]
  • Sieh M, Bolen JB, Weiss A. CD45 specifically modulates binding of Lck to a phosphopeptide encompassing the negative regulatory tyrosine of Lck. EMBO J. 1993 Jan;12(1):315–321. [PubMed]
  • Slilaty SN, Fung M, Shen SH, Lebel S. Site-directed mutagenesis by complementary-strand synthesis using a closing oligonucleotide and double-stranded DNA templates. Anal Biochem. 1990 Feb 15;185(1):194–200. [PubMed]
  • Stanners CP, Eliceiri GL, Green H. Two types of ribosome in mouse-hamster hybrid cells. Nat New Biol. 1971 Mar 10;230(10):52–54. [PubMed]
  • Sudol M, Greulich H, Newman L, Sarkar A, Sukegawa J, Yamamoto T. A novel Yes-related kinase, Yrk, is expressed at elevated levels in neural and hematopoietic tissues. Oncogene. 1993 Apr;8(4):823–831. [PubMed]
  • Thomas ML, Reynolds PJ, Chain A, Ben-Neriah Y, Trowbridge IS. B-cell variant of mouse T200 (Ly-5): evidence for alternative mRNA splicing. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5360–5363. [PubMed]
  • Trowbridge IS, Ostergaard HL, Johnson P. CD45: a leukocyte-specific member of the protein tyrosine phosphatase family. Biochim Biophys Acta. 1991 Oct 16;1095(1):46–56. [PubMed]
  • Veillette A, Abraham N, Caron L, Davidson D. The lymphocyte-specific tyrosine protein kinase p56lck. Semin Immunol. 1991 May;3(3):143–152. [PubMed]
  • Veillette A, Bolen JB, Bookman MA. Alterations in tyrosine protein phosphorylation induced by antibody-mediated cross-linking of the CD4 receptor of T lymphocytes. Mol Cell Biol. 1989 Oct;9(10):4441–4446. [PMC free article] [PubMed]
  • Veillette A, Bookman MA, Horak EM, Bolen JB. The CD4 and CD8 T cell surface antigens are associated with the internal membrane tyrosine-protein kinase p56lck. Cell. 1988 Oct 21;55(2):301–308. [PubMed]
  • Veillette A, Bookman MA, Horak EM, Samelson LE, Bolen JB. Signal transduction through the CD4 receptor involves the activation of the internal membrane tyrosine-protein kinase p56lck. Nature. 1989 Mar 16;338(6212):257–259. [PubMed]
  • Veillette A, Caron L, Fournel M, Pawson T. Regulation of the enzymatic function of the lymphocyte-specific tyrosine protein kinase p56lck by the non-catalytic SH2 and SH3 domains. Oncogene. 1992 May;7(5):971–980. [PubMed]
  • Veillette A, Davidson D. Src-related protein tyrosine kinases and T-cell receptor signalling. Trends Genet. 1992 Feb;8(2):61–66. [PubMed]
  • Veillette A, Horak ID, Bolen JB. Post-translational alterations of the tyrosine kinase p56lck in response to activators of protein kinase C. Oncogene Res. 1988 May;2(4):385–401. [PubMed]
  • Winkler DG, Park I, Kim T, Payne NS, Walsh CT, Strominger JL, Shin J. Phosphorylation of Ser-42 and Ser-59 in the N-terminal region of the tyrosine kinase p56lck. Proc Natl Acad Sci U S A. 1993 Jun 1;90(11):5176–5180. [PubMed]
  • Zheng XM, Wang Y, Pallen CJ. Cell transformation and activation of pp60c-src by overexpression of a protein tyrosine phosphatase. Nature. 1992 Sep 24;359(6393):336–339. [PubMed]

Articles from Molecular and Cellular Biology are provided here courtesy of American Society for Microbiology (ASM)