Abstract
The actin filament-associated protein AFAP-110 forms a stable complex with activated variants of Src in chick embryo fibroblast cells. Stable complex formation requires the integrity of the Src SH2 and SH3 domains. In addition, AFAP-110 encodes two adjacent SH3 binding motifs and six candidate SH2 binding motifs. These data indicate that both SH2 and SH3 domains may work cooperatively to facilitate Src/AFAP-110 stable complex formation. As a test for this hypothesis, we sought to understand whether one or both SH3 binding motifs in AFAP-110 modulate interactions with the Src SH3 domain and if this interaction was required to present AFAP-110 for tyrosine phosphorylation by, and stable complex formation with, Src. A proline to alanine site-directed mutation in the amino terminal SH3 binding motif (SH3bm I) was sufficient to abrogate absorption of AFAP-110 with GST-SH3src. Co-expression of activated Src (pp60527F) with AFAP-110 in Cos-1 cells permit tyrosine phosphorylation of AFAP-110 a nd stable complex formation with pp60527F. However, co-expression of the SH3 null-binding mutant (AFAP71A) with pp60527F revealed a 2.7 fold decrease in steady-state levels of tyrosine phosphorylation, compared to AFAP-110. Although a lower but detectable level of AFAP71A was phosphorylated on tyrosine, AFAP71A could not be detected in stable complex with pp60527F, unlike AFAP-110. These data indicate that SH3 interactions facilitate presentation of AFAP-110 for tyrosine phosphorylation and are also required for stable complex formation with pp60527F. (Mol Cell Biochem 175: 243–252, 1997)
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References
Cooper JA, Brown MT: Regulation, substrates and functions of src. Biochim Biophys Acta Gene Struct Exper 1287: 121–149, 1996
Barnekow A, Paul E, Schartl M: Expression of the c-src protooncogene in human skin tumors. Cancer Res 47: 235–240, 1987
Bolen JB, Veillette A, Schwartz AM, DeSeau V, Rosen N: Activation of pp60c-src protein kinase activity in human colon carcinoma. Proc Natl Acad Sci USA 84: 2251–2255, 1987
Cartwright CA, Kamps MP, Meisler AI, Pipas JM, Ekhart W: pp60c-src activation in human colon carcinoma. J Clin Invest 83: 2025–2033, 1989
Cartwright CA, Meisler AI, Eckhart W: Activation of the pp60 c-src protein kinase is an early event in colonic carcinogenesis. Proc Natl Acad Sci USA 87: 558–562, 1990
Jacobs C, Rubsamen H: Expression of pp60 c-src protein kinase in adult and fetal human tissues: High activities in some sarcomas and mammary carcinomas. Cancer Res 43: 1696–1702, 1983
Rosen N, Bolen JB, Schwartz AM, Cohen P, DeSeau V, Isreal MA: Analysis of pp60 c-src protein kinase activity in human tumor cell lines and tissues. J Biol Chem 261: 13754–13759, 1986
Schaller MD, Bouton AH, Flynn DC, Parsons JT: In: WE Cohn, K Moldave (eds). Progress in Nucleic Acid Research and Molecular Biology. Academic Press Inc., San Diego, CA, 1993 44: 205–227
Pawson T: Protein modules and signalling networks. Nature 373: 573–580, 1995
Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, Neel BG, Birge RB, Fajardo JE, Chou MM, Hanatusa H, Schaffhausen B, Cantley LC: SH2 domains recognize specific phosphopeptide sequences. Cell 72: 767–778, 1993
Ren R, Mayer BJ, Cicchetti P, Baltimore D: Identification of a tenamino acid proline-rich SH3 binding site. Science 259: 1157–1161, 1993
Yu H, Rosen MK, Shin TB, Seidel-Dugan C, Brugge JS, Schreiber SL: Solution structure of the SH3 domain of Src and Identification of its ligand-binding site. Science 258: 1665–1670, 1993
Lemmon MA, Ladbury JE, Mandiyan V, Zhou M, Schlessinger J: Independent binding of peptide ligands to the SH2 and SH3 domains of Grb2. J Biol Chem 269: 31653–31658, 1994
Flynn DC, Horne T-L, Reynolds AB, Parsons JT: Identification and sequence analysis of cDNAs encoding a 110 kilodalton actin filament associated pp60 src substrate. Mol Cell Biol 13: 7892–7900, 1993
Brott BK, Decker S, O'Brien MC, Jove R: Molecular features of the viral and cellular Src kinases involved in interactions with the GTPaseactivating protein. Mol Cell Biol 11: 5059–5067, 1991
Fumagalli S, Totty NF, Hsuan JJ, Courtneidge SA: A target for Src in mitosis. Nature 368: 871–874, 1994
Taylor SJ, Shalloway D: An RNA-binding protein associated with Src through its SH2 and SH3 domains in mitosis. Nature 368: 867–871, 1994
Turner CE, Miller JT: Primary sequence of paxillin contains putative SH2 and SH3 domain binding motifs and multiple LIM domains: Identification of a vinculin and pp125Fak-binding region. J Cell Sci 107: 1583–1591, 1993
Cobb BS, Schaller MD, Leu T-H, Parsons JT: Stable association of pp60src and pp59fyn with focal adhesion-associated protein tyrosine kinase, pp125 Fak Mol Cell Biol 14: 147–155, 1994
Schaller MD, Hildebrand JD, Shannon JD, Fox FW, Vines RR, Parsons JT: Autophosphorylation of the focal adhesion kinase pp125 Fak, directs SH2-dependent binding of pp60 src. Mol Cell Biol 14: 1680–1688, 1994
Kanner SB, Reynolds AB, Wang, H-C, Parsons JT: The SH2 and SH3 domains of pp60 src direct stable association with tyrosine phosphorylated proteins p130 and p110. EMBO J 10: 1689–1698, 1991
Reynolds AB, Kanner SB, Wang H-C, Parsons JT: Stable association of activated pp60 src with two tyrosine phosphorylated cellular proteins. Mol Cell Biol 9: 3951–3958, 1989
Sakai R, Iwamatsu A, Hirano N, Ogawa S, Tanaka T, Mano H, Yazaki Y, Hirai H: A novel signalling molecule, p130, forms stable complexes in vivo with v-Crk and v-Src in a tyrosine phosphorylation-dependent manner. EMBO J 13: 3748–3756, 1994
Weng A, Thomas SM, Rickles RJ, Taylor JA, Brauer AW, Seidel-Dugan C, Michael WM, Dreyfuss G, Brugge JS: Identification of Src, Fyn, and Lyn SH3-binding proteins: Implications for a function of SH3 domains. Mol Cell Biol 14: 4509–4521, 1994
Weng Z, Rickles RJ, Feng S, Richard S, Shaw AS, Schrieiber SL, Brugge JS: Structure function analysis of SH3 domains: SH3 binding specificity altered by single amino acid substitutions. Mol Cell Biol 15: 5627–5634, 1995
Liu X, Marengere LE, Koch CA, Pawson T: The v-Src SH3 domain binds phosphatidylinositol 3'-kinase. Mol Cell Biol 13: 5225–5232, 1993
Prasad KV, Kapeller R, Janssen O, Repke H, Duke-Cohan JS, Cantley LC, Rudd CE: Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p561ck complex: The p561ck SH3 domain binds to PI 3-kinase but not PI 4-kinase. Mol Cell Biol 13: 7708–7717, 1993
Vogel LB, Fujita DJ: The SH3 domain of p561ck is involved in binding to phosphatidylinositol 3'-kinase from T lymphocytes. Mol Cell Biol 13: 7408–7417, 1993
Wages DS, Deefer J, Rall TB, and Weber MJ: Mutations in the SH3 domain of the src ongene which decrease association of phosphatidylinositol 3'-kinase activity with pp60v-src and alter cellular morphology. J Virol 66: 1866–1874, 1992
Gout I, Dhand R, Hiles ID, Fry MJ, Panayotou G, Das P, Truong O, Totty NF, Hsuan J, Booker GW, Campbell ID, Waterfield MD: The GTPase dynamin binds to and is activated by a subset of SH3 domains. Cell 75: 25–36, 1993
Okada M, Howel BW, Broome MA, Cooper JA: Deletion of the SH3 domain of Src interferes with regulation by the phosphorylated carboxyl-terminal tyrosine. J Biol Chem 258: 18070–18075, 1993
Potts WM, Reynolds AB, Lansing TJ, Parsons JT: Activation of pp60 csrc transforming potential by mutations altering the structure of an amino terminal domain containing residues 90–95. Oncogene Res 3: 343–355, 1988
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 12: 1835–1845, 1992
Superti-Furga G, Fumafalli S, Doegl M, Courtneidge SA, Draaetta G: Csk inhibition of c-Src activity requires both the SH2 and SH3 domains. EMBO J 12: 2625–2634, 1993
Panchamoorthy G, Fukazawa T, Stolz L, Payne G, Reedquist K, Shoelson S, Zhou S, Cantley L, Walsh C, Band H: Physical and functional interactions between SH2 and SH3 domains of the Src family protein tyrosine kinase p59fyn. Mol Cell Biol 14: 6372–6385, 1994
Flynn DC, Koay TC, Humphries CG, Guappone AC: AFAP-120: A variant form of the Src SH2/SH3 binding partner AFAP-110 is detected in brain and contains a novel internal sequence which binds to a 67 kDa protein. J Biol Chem 270: 3894–3899, 1995
Bar-Sagi D, Rotin D, Batzer A, Mandiyan V, Schlessinger J: SH3 domains direct cellular localization of signalling molecules. Cell 74: 83–91, 1993
Felice GR, Eason P, Nermut MV, Kellie S: Pp60 v-src association with the cytoskeleton induces actin reorganization without affecting polymerization status. Eur J Cell Biol 52: 4759, 1990
Reynolds AB, Roesel DJ, Kanner SB, Parsons JT: Transformationspecific tyrosine phosphorylation of a novel cellular protein in chicken cells expressing oncogenic variants of the avian cellular Src gene. Mol Cell Biol 9: 629–638, 1989
Guappone AC, Qian Y, Weimer TG, Flynn DC: An in vivo system for analysis of stable complex formation between Src and AFAP-110. Meth Cell Sci 18: 1–11, 1996
Chen C, Okayama H: High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol 7: 2745–2752, 1987
Flynn DC, Schaller MD, Parsons JT: Tyrosine phosphorylation of a 120,000 Dalton membrane-associated protein by the neural form of pp60 c-src, pp60 c-src+. Oncogene 7: 579–583, 1992
Smith DB, Johnson KS: Single-step purification of polypeptides expressed in Escherichia coli as fusions with gultathione S-transferase. Gene 67: 31–40, 1988
Parsons SJ, McCarley DJ, Raymond VW, Parsons JT: Monoclonal antibodies to Rous sarcoma virus pp60 src react with enzymatically active cellular pp60 src of avian and mammalian origin. J Virol 51: 272–282, 1984
Cheadle C, Ivashchenko Y, South V, Searfoss GH, French S, Howk R, Ricca GA, Jaye M: Identification of a Src SH3 domain binding motif by screening a random phage display library. J Biol Chem 269: 24034–24039, 1994
Rickles RJ, Botfield MC, Weng Z, Taylor JA, Green OM, Brugge JS, Zoller MJ: Identification of Src, Fyn, Lyn, PI3-K and Abl SH3 domain ligands using phage display libraries. EMBO J 13: 5598–5604, 1994
Rickles RJ, Botfield MC, Zhou X-M, Henry PA, Brugge JS, Zoller MJ: Phage display selection of ligand residues important for Src homology 3 domain binding specificity. Proc Natl Acad Sci USA 92: 10909–10913, 1995
Sparks AB, Quilliam LA, Thorn JM, Der CJ, Kay BK: Identification and characterization of Src SH3 ligands from phage-displayed random peptide libraries. J Biol Chem 269: 23853–23856, 1994
Hamaguchi M, Hanafusa H: Association of p60 src with Triton X-100-resistant cellular structure correlates with morphological transformation. Proc Natl Acad Sci USA 84: 2312–2316, 1987
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Guappone, A.C., Flynn, D.C. The integrity of the SH3 binding motif of AFAP-110 is required to facilitate tyrosine phosphorylation by, and stable complex formation with, Src. Mol Cell Biochem 175, 243–252 (1997). https://doi.org/10.1023/A:1006840104666
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DOI: https://doi.org/10.1023/A:1006840104666