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. 1992 Oct 15;89(20):9559–9563. doi: 10.1073/pnas.89.20.9559

Direct analysis of the binding of Src-homology 2 domains of phospholipase C to the activated epidermal growth factor receptor.

G Zhu 1, S J Decker 1, A R Saltiel 1
PMCID: PMC50171  PMID: 1384057

Abstract

A number of proteins involved in intracellular signaling contain regions of homology to the product of the src oncogene that are termed Src-homology (SH) 2 domains. SH2 domains are believed to mediate the association of these proteins with various tyrosine-phosphorylated receptors in a growth factor-dependent manner. We have examined the kinetic characteristics of one of these interactions, the binding of the SH2 domains of phospholipase C gamma 1 with the receptor for epidermal growth factor (EGF). Bacterial fusion proteins were prepared containing the two SH2 domains of PLC gamma 1 and labeled metabolically with [35S]methionine/cysteine. A fusion protein containing both SH2 domains bound to the purified EGF receptor from EGF-treated cells, whereas no binding to receptors from control cells was detected. Binding was rapid, reaching apparent equilibrium by 10 min. Dissociation of the complex occurred only in the presence of excess unlabeled SH2 protein and exhibited two kinetic components. Similarly, analysis of apparent equilibrium binding revealed a nonlinear Scatchard plot, further indicating complex binding kinetics that may reflect cooperative behavior. The binding of the fusion protein containing both SH2 domains was inhibited by a fusion protein containing only the amino-terminal SH2 domain, although at concentrations an order of magnitude higher than that observed with the complete fusion protein. Fusion proteins containing SH2 domains from the GTPase-activating protein, the p85 regulatory subunit of phosphatidylinositol 3'-kinase, or the Abl oncoprotein competed less effectively. Binding of the PLC gamma 1 SH2 fusion protein to a mutant EGF receptor lacking the two carboxyl-terminal tyrosine phosphorylation sites exhibited a significantly lower affinity than that observed with the wild type, suggesting that this region of the receptor may play an important role. This binding assay represents a means with which to evaluate the pleiotropic nature of growth factor action.

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Selected References

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  1. Anderson D., Koch C. A., Grey L., Ellis C., Moran M. F., Pawson T. Binding of SH2 domains of phospholipase C gamma 1, GAP, and Src to activated growth factor receptors. Science. 1990 Nov 16;250(4983):979–982. doi: 10.1126/science.2173144. [DOI] [PubMed] [Google Scholar]
  2. Auger K. R., Serunian L. A., Soltoff S. P., Libby P., Cantley L. C. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989 Apr 7;57(1):167–175. doi: 10.1016/0092-8674(89)90182-7. [DOI] [PubMed] [Google Scholar]
  3. Backer J. M., Schroeder G. G., Kahn C. R., Myers M. G., Jr, Wilden P. A., Cahill D. A., White M. F. Insulin stimulation of phosphatidylinositol 3-kinase activity maps to insulin receptor regions required for endogenous substrate phosphorylation. J Biol Chem. 1992 Jan 15;267(2):1367–1374. [PubMed] [Google Scholar]
  4. Berridge M. J., Heslop J. P., Irvine R. F., Brown K. D. Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor. Biochem J. 1984 Aug 15;222(1):195–201. doi: 10.1042/bj2220195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Besterman J. M., Watson S. P., Cuatrecasas P. Lack of association of epidermal growth factor-, insulin-, and serum-induced mitogenesis with stimulation of phosphoinositide degradation in BALB/c 3T3 fibroblasts. J Biol Chem. 1986 Jan 15;261(2):723–727. [PubMed] [Google Scholar]
  6. Bustelo X. R., Ledbetter J. A., Barbacid M. Product of vav proto-oncogene defines a new class of tyrosine protein kinase substrates. Nature. 1992 Mar 5;356(6364):68–71. doi: 10.1038/356068a0. [DOI] [PubMed] [Google Scholar]
  7. Chen W. S., Lazar C. S., Lund K. A., Welsh J. B., Chang C. P., Walton G. M., Der C. J., Wiley H. S., Gill G. N., Rosenfeld M. G. Functional independence of the epidermal growth factor receptor from a domain required for ligand-induced internalization and calcium regulation. Cell. 1989 Oct 6;59(1):33–43. doi: 10.1016/0092-8674(89)90867-2. [DOI] [PubMed] [Google Scholar]
  8. Coughlin S. R., Escobedo J. A., Williams L. T. Role of phosphatidylinositol kinase in PDGF receptor signal transduction. Science. 1989 Mar 3;243(4895):1191–1194. doi: 10.1126/science.2466336. [DOI] [PubMed] [Google Scholar]
  9. Decker S. J., Alexander C., Habib T. Epidermal growth factor (EGF)-stimulated tyrosine phosphorylation and EGF receptor degradation in cells expressing EGF receptors truncated at residue 973. J Biol Chem. 1992 Jan 15;267(2):1104–1108. [PubMed] [Google Scholar]
  10. Downing J. R., Margolis B. L., Zilberstein A., Ashmun R. A., Ullrich A., Sherr C. J., Schlessinger J. Phospholipase C-gamma, a substrate for PDGF receptor kinase, is not phosphorylated on tyrosine during the mitogenic response to CSF-1. EMBO J. 1989 Nov;8(11):3345–3350. doi: 10.1002/j.1460-2075.1989.tb08496.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Escobedo J. A., Kaplan D. R., Kavanaugh W. M., Turck C. W., Williams L. T. A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine. Mol Cell Biol. 1991 Feb;11(2):1125–1132. doi: 10.1128/mcb.11.2.1125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Escobedo J. A., Navankasattusas S., Kavanaugh W. M., Milfay D., Fried V. A., Williams L. T. cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor. Cell. 1991 Apr 5;65(1):75–82. doi: 10.1016/0092-8674(91)90409-r. [DOI] [PubMed] [Google Scholar]
  13. Guan K. L., Dixon J. E. Eukaryotic proteins expressed in Escherichia coli: an improved thrombin cleavage and purification procedure of fusion proteins with glutathione S-transferase. Anal Biochem. 1991 Feb 1;192(2):262–267. doi: 10.1016/0003-2697(91)90534-z. [DOI] [PubMed] [Google Scholar]
  14. Johnson R. M., Garrison J. C. Epidermal growth factor and angiotensin II stimulate formation of inositol 1,4,5- and inositol 1,3,4-trisphosphate in hepatocytes. Differential inhibition by pertussis toxin and phorbol 12-myristate 13-acetate. J Biol Chem. 1987 Dec 25;262(36):17285–17293. [PubMed] [Google Scholar]
  15. Kaplan D. R., Morrison D. K., Wong G., McCormick F., Williams L. T. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990 Apr 6;61(1):125–133. doi: 10.1016/0092-8674(90)90220-9. [DOI] [PubMed] [Google Scholar]
  16. Kazlauskas A., Cooper J. A. Phosphorylation of the PDGF receptor beta subunit creates a tight binding site for phosphatidylinositol 3 kinase. EMBO J. 1990 Oct;9(10):3279–3286. doi: 10.1002/j.1460-2075.1990.tb07527.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Kazlauskas A., Ellis C., Pawson T., Cooper J. A. Binding of GAP to activated PDGF receptors. Science. 1990 Mar 30;247(4950):1578–1581. doi: 10.1126/science.2157284. [DOI] [PubMed] [Google Scholar]
  18. Kim H. K., Kim J. W., Zilberstein A., Margolis B., Kim J. G., Schlessinger J., Rhee S. G. PDGF stimulation of inositol phospholipid hydrolysis requires PLC-gamma 1 phosphorylation on tyrosine residues 783 and 1254. Cell. 1991 May 3;65(3):435–441. doi: 10.1016/0092-8674(91)90461-7. [DOI] [PubMed] [Google Scholar]
  19. Kumjian D. A., Wahl M. I., Rhee S. G., Daniel T. O. Platelet-derived growth factor (PDGF) binding promotes physical association of PDGF receptor with phospholipase C. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8232–8236. doi: 10.1073/pnas.86.21.8232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Lev S., Givol D., Yarden Y. Interkinase domain of kit contains the binding site for phosphatidylinositol 3' kinase. Proc Natl Acad Sci U S A. 1992 Jan 15;89(2):678–682. doi: 10.1073/pnas.89.2.678. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Margolis B. L., Lax I., Kris R., Dombalagian M., Honegger A. M., Howk R., Givol D., Ullrich A., Schlessinger J. All autophosphorylation sites of epidermal growth factor (EGF) receptor and HER2/neu are located in their carboxyl-terminal tails. Identification of a novel site in EGF receptor. J Biol Chem. 1989 Jun 25;264(18):10667–10671. [PubMed] [Google Scholar]
  22. Margolis B., Bellot F., Honegger A. M., Ullrich A., Schlessinger J., Zilberstein A. Tyrosine kinase activity is essential for the association of phospholipase C-gamma with the epidermal growth factor receptor. Mol Cell Biol. 1990 Feb;10(2):435–441. doi: 10.1128/mcb.10.2.435. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Margolis B., Li N., Koch A., Mohammadi M., Hurwitz D. R., Zilberstein A., Ullrich A., Pawson T., Schlessinger J. The tyrosine phosphorylated carboxyterminus of the EGF receptor is a binding site for GAP and PLC-gamma. EMBO J. 1990 Dec;9(13):4375–4380. doi: 10.1002/j.1460-2075.1990.tb07887.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Margolis B., Rhee S. G., Felder S., Mervic M., Lyall R., Levitzki A., Ullrich A., Zilberstein A., Schlessinger J. EGF induces tyrosine phosphorylation of phospholipase C-II: a potential mechanism for EGF receptor signaling. Cell. 1989 Jun 30;57(7):1101–1107. doi: 10.1016/0092-8674(89)90047-0. [DOI] [PubMed] [Google Scholar]
  25. Matsuda M., Mayer B. J., Fukui Y., Hanafusa H. Binding of transforming protein, P47gag-crk, to a broad range of phosphotyrosine-containing proteins. Science. 1990 Jun 22;248(4962):1537–1539. doi: 10.1126/science.1694307. [DOI] [PubMed] [Google Scholar]
  26. Mayer B. J., Hamaguchi M., Hanafusa H. A novel viral oncogene with structural similarity to phospholipase C. Nature. 1988 Mar 17;332(6161):272–275. doi: 10.1038/332272a0. [DOI] [PubMed] [Google Scholar]
  27. Mayer B. J., Hanafusa H. Association of the v-crk oncogene product with phosphotyrosine-containing proteins and protein kinase activity. Proc Natl Acad Sci U S A. 1990 Apr;87(7):2638–2642. doi: 10.1073/pnas.87.7.2638. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Mayer B. J., Jackson P. K., Baltimore D. The noncatalytic src homology region 2 segment of abl tyrosine kinase binds to tyrosine-phosphorylated cellular proteins with high affinity. Proc Natl Acad Sci U S A. 1991 Jan 15;88(2):627–631. doi: 10.1073/pnas.88.2.627. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Meisenhelder J., Suh P. G., Rhee S. G., Hunter T. Phospholipase C-gamma is a substrate for the PDGF and EGF receptor protein-tyrosine kinases in vivo and in vitro. Cell. 1989 Jun 30;57(7):1109–1122. doi: 10.1016/0092-8674(89)90048-2. [DOI] [PubMed] [Google Scholar]
  30. Molloy C. J., Bottaro D. P., Fleming T. P., Marshall M. S., Gibbs J. B., Aaronson S. A. PDGF induction of tyrosine phosphorylation of GTPase activating protein. Nature. 1989 Dec 7;342(6250):711–714. doi: 10.1038/342711a0. [DOI] [PubMed] [Google Scholar]
  31. Moran M. F., Koch C. A., Anderson D., Ellis C., England L., Martin G. S., Pawson T. Src homology region 2 domains direct protein-protein interactions in signal transduction. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8622–8626. doi: 10.1073/pnas.87.21.8622. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Ohmichi M., Decker S. J., Pang L., Saltiel A. R. Nerve growth factor binds to the 140 kd trk proto-oncogene product and stimulates its association with the src homology domain of phospholipase C gamma 1. Biochem Biophys Res Commun. 1991 Aug 30;179(1):217–223. doi: 10.1016/0006-291x(91)91357-i. [DOI] [PubMed] [Google Scholar]
  33. Ohmichi M., Decker S. J., Pang L., Saltiel A. R. Phospholipase C-gamma 1 directly associates with the p70 trk oncogene product through its src homology domains. J Biol Chem. 1991 Aug 15;266(23):14858–14861. [PubMed] [Google Scholar]
  34. Otsu M., Hiles I., Gout I., Fry M. J., Ruiz-Larrea F., Panayotou G., Thompson A., Dhand R., Hsuan J., Totty N. Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. Cell. 1991 Apr 5;65(1):91–104. doi: 10.1016/0092-8674(91)90411-q. [DOI] [PubMed] [Google Scholar]
  35. Pike L. J., Eakes A. T. Epidermal growth factor stimulates the production of phosphatidylinositol monophosphate and the breakdown of polyphosphoinositides in A431 cells. J Biol Chem. 1987 Feb 5;262(4):1644–1651. [PubMed] [Google Scholar]
  36. Reedijk M., Liu X. Q., Pawson T. Interactions of phosphatidylinositol kinase, GTPase-activating protein (GAP), and GAP-associated proteins with the colony-stimulating factor 1 receptor. Mol Cell Biol. 1990 Nov;10(11):5601–5608. doi: 10.1128/mcb.10.11.5601. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Rotin D., Margolis B., Mohammadi M., Daly R. J., Daum G., Li N., Fischer E. H., Burgess W. H., 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. doi: 10.1002/j.1460-2075.1992.tb05087.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Stahl M. L., Ferenz C. R., Kelleher K. L., Kriz R. W., Knopf J. L. Sequence similarity of phospholipase C with the non-catalytic region of src. Nature. 1988 Mar 17;332(6161):269–272. doi: 10.1038/332269a0. [DOI] [PubMed] [Google Scholar]
  39. Suh P. G., Ryu S. H., Moon K. H., Suh H. W., Rhee S. G. Inositol phospholipid-specific phospholipase C: complete cDNA and protein sequences and sequence homology to tyrosine kinase-related oncogene products. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5419–5423. doi: 10.1073/pnas.85.15.5419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Todderud G., Wahl M. I., Rhee S. G., Carpenter G. Stimulation of phospholipase C-gamma 1 membrane association by epidermal growth factor. Science. 1990 Jul 20;249(4966):296–298. doi: 10.1126/science.2374928. [DOI] [PubMed] [Google Scholar]
  41. Trahey M., Wong G., Halenbeck R., Rubinfeld B., Martin G. A., Ladner M., Long C. M., Crosier W. J., Watt K., Koths K. Molecular cloning of two types of GAP complementary DNA from human placenta. Science. 1988 Dec 23;242(4886):1697–1700. doi: 10.1126/science.3201259. [DOI] [PubMed] [Google Scholar]
  42. Varticovski L., Druker B., Morrison D., Cantley L., Roberts T. The colony stimulating factor-1 receptor associates with and activates phosphatidylinositol-3 kinase. Nature. 1989 Dec 7;342(6250):699–702. doi: 10.1038/342699a0. [DOI] [PubMed] [Google Scholar]
  43. Vega Q. C., Cochet C., Filhol O., Chang C. P., Rhee S. G., Gill G. N. A site of tyrosine phosphorylation in the C terminus of the epidermal growth factor receptor is required to activate phospholipase C. Mol Cell Biol. 1992 Jan;12(1):128–135. doi: 10.1128/mcb.12.1.128. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Velu T. J., Vass W. C., Lowy D. R., Beguinot L. Functional heterogeneity of proto-oncogene tyrosine kinases: the C terminus of the human epidermal growth factor receptor facilitates cell proliferation. Mol Cell Biol. 1989 Apr;9(4):1772–1778. doi: 10.1128/mcb.9.4.1772. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Vogel U. S., Dixon R. A., Schaber M. D., Diehl R. E., Marshall M. S., Scolnick E. M., Sigal I. S., Gibbs J. B. Cloning of bovine GAP and its interaction with oncogenic ras p21. Nature. 1988 Sep 1;335(6185):90–93. doi: 10.1038/335090a0. [DOI] [PubMed] [Google Scholar]
  46. Wahl M. I., Daniel T. O., Carpenter G. Antiphosphotyrosine recovery of phospholipase C activity after EGF treatment of A-431 cells. Science. 1988 Aug 19;241(4868):968–970. doi: 10.1126/science.2457254. [DOI] [PubMed] [Google Scholar]
  47. Yi T. L., Cleveland J. L., Ihle J. N. Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13. Mol Cell Biol. 1992 Feb;12(2):836–846. doi: 10.1128/mcb.12.2.836. [DOI] [PMC free article] [PubMed] [Google Scholar]

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