Stable association of activated pp60src with two tyrosine-phosphorylated cellular proteins.

Abstract

We have identified two phosphotyrosine-containing cellular proteins with relative molecular masses of 130,000 (ppl30) and 110,000 (ppllO) daltons in chicken embryo cells that coimmunoprecipitated with pp60v-src and activated forms of chicken pp6ocsrc (pp60527F). Most if not all of the tyrosine-phosphorylated forms of ppl30 and ppllO could be immunoprecipitated from lysates with any of several src protein-specific monoclonal antibodies directed against at least three spatially distinct epitopes. Consequently, of the more than 15 prominent phosphoproteins detected on immunoblots with phosphotyrosine-specific antibodies, ppl30 and ppl1O were selectively removed by src protein-specific immunoprecipitation, and their presence in the immunoprecipitates appears to have been due to a direct interaction with activated src proteins. src protein variants that induce different morphological phenotypes were altered in their ability to form detergent-stable complexes with ppl30 and ppllO or with ppllO alone. Mutant src proteins, defective for myristylation, showed increased tyrosine phosphorylation of and association with ppllO. Expression of src variants with mutations in the A box (pp60dl921527F) or B box (pp6OdIl55/527F) of the src homology region induced differences in phosphorylation of ppl30 and pplO, as well as changes in their association with variant src proteins. Sequences within the B-box region appeared to be necessary for stable complex formation with pp130 and ppllO and may be involved in the interaction of activated src proteins with cellular substrates.The transforming gene of Rous sarcoma virus (RSV), v-src, encodes a 60-kilodalton (kDa) protein tyrosine kinase (pp60vsr() whose expression is both necessary and sufficient to initiate and maintain cellular transformation (1, 2, 19-21, 23, 35, 56; J. A. Cooper, in B. Kemp and P. F. Alewood, ed., Peptides and Protein PhosphoylWation, in press). Its normal cellular counterpart, c-src, encodes a similar protein (pp6Oc--') which is not oncogenic, even when expressed at high levels (22,41,57). Although there are numerous amino acid differences between the two proteins, the major oncogenic difference occurs at the C-terminal end, where 19 C-terminal amino acids in pp6Ocsr( have been replaced with 12 unrelated amino acids in pp60v-r'c (55,60). The kinase activity of pp60c--"' is restricted by phosphorylation of a C-terminal tyrosine residue, Tyr-527, which is not present in pp60vs"" (10,11,13,34). Mutations within this C-terminal regulatory region, e.g., Tyr-527 to Phe or Ser or the truncation of the c-src protein within the C-terminal 17 residues, activate the tyrosine protein kinase activity of pp6Oc-''' and concomitantly, the transforming potential of the c-src gene product (8,30,31,47,52).Other structural domains within pp60''' have been shown to influence transforming activity. Myristylation is required for stable membrane association and transformation (6,7,14,16,54). Substitution of Ala or Glu for Gly-2 yields a soluble src protein with unaltered tyrosine kina...

The intrinsic ability of AFAP-110 to alter actin filament integrity is linked with its ability to also activate cellular tyrosine kinases

“…One possible mechanism by which AFAP-110 Dlzip could activate tyrosine kinases like cSrc might be through its ability to function as an SH2/SH3 binding partner (Flynn et al, 1993;Guappone et al, 1998;Guappone and Flynn, 1997;Kanner et al, 1991;Reynolds et al, 1989). It has been shown the SH3 binding partners, such as the HIV encoded gene product, Nef, can activate the Src family member Hck via SH3 binding (Briggs et al, 1997;Moare® et al, 1997).…”

“…Activation of the Src oncogene is associated with characteristic changes in the actin cytoskeleton of cultured cells (Felice et al, 1990;Hamaguchi and Hanafusa, 1987;Reynolds et al, 1989). These changes include a repositioning of actin ®laments into rosettelike structures, and the formation of lamellipodia and ®lopodia, which are associated with increased cell motility and the onset of tumor metastasis in humans (Bolen et al, 1987;Boschek et al, 1981;Cartwright et al, 1990;Irby et al, 1999;Rosen et al, 1986;Tarone et al, 1985).…”

Src Points the Way to Biomarkers and Chemotherapeutic Targets

“…As its name denotes, Cas associates with Crk (CT10 regulator of kinase), a linker protein containing SH2 and SH3 domains. [37][38][39] Src, Cas, and Crk work together to regulate cell adhesion, migration, and morphological changes associated with neoplastic transformation. 23,40,41 After immunoaffinity purification of Cas from cells expressing v-Crk, Sakai et al 42 isolated the cDNA for Cas and confirmed that it served as an efficient Src kinase substrate.…”

Processive phosphorylation: Mechanism and biological importance

“…p130Cas (Crk-associated substrate, 130kDa) was first identified as a protein that is highly tyrosine phosphorylated in v-Src and v-Crk transformed cells and that forms stable complexes with these oncoproteins [78][79][80]. Cas is a multidomain protein containing an N-terminal SH3 domain and Pro-rich sequences, a large central substrate domain with 15 repeats of the motif YXXP, and a C-terminal Src binding region [81] (Figure 4).…”