Deletions in the SH2 domain of p60v-src prevent association with the detergent-insoluble cellular matrix.

Abstract

p60v-src has been shown to associate with a detergent-insoluble cellular matrix containing cytoskeletal proteins, but p60csrc does not bind to this matrix. We analyzed the association of mutant src proteins with the matrix and found that mutants which lack an amino-terminal portion (residues 149 to 169) of the SH2 domain cannot bind to the matrix. Neither the SH3 region nor other portions of the SH2 region were required for association. We also tested protein kinase-defective mutants and chimeras of p6O-src and p60csrc. We found a strong correlation between the kinase activity of p6Osrc and its association with the detergent-insoluble matrix. Double infection of kinase-defective and kinase-active mutants did not result in matrix binding of the kinase-defective src proteins. We also found that Tyr-416, the major site of autophosphorylation in p60vsrc, was not required for matrix association.Rous sarcoma virus (RSV) encodes the oncogene v-src. The product of v-src, p60vsrc, is a tyrosine-specific protein kinase (19). p60vsrc is myristoylated on a glycine residue at the amino terminus and localizes in the membrane fraction (19). A number of cellular proteins are phosphorylated on tyrosine upon transformation with p60vsrc. Most of these are also in the membrane fraction (13), suggesting that specific localization of p60v-src is important for cell transformation.Burr et al. (3) have demonstrated association of p60v-src with a detergent-resistant subcellular structure that consists mostly of cytoskeleton. It has been previously reported that most transforming variants of p60vsrc associate with this structure but that nontransforming variants, including the cellular proto-oncogene p60csrc, do not (12,30). However, it is not known which sequences of p60v-src are necessary for its association with this structure.Two regions of sequence conservation have been identified in the amino termini of cytoplasmic tyrosine kinases, including p6Osrc (32, 36). These regions are SH2 (src homology 2, residues 137 through 241 in src; see Fig. lA) and SH3 (src homology 3, residues 84 through 114). SH2 sequences are also present in phospholipase C--y (39), GAP (ras GTPase-activating protein) (43), and the crk oncogene product (32). Mutations of the SH2 regions of v-src and v-fps produce transformation-defective or temperature-sensitive tyrosine kinase proteins (2,5,25,26,36,44). Point mutations in the SH2 region of the proto-oncogene p60c-src have been shown to elevate kinase activity and transforming ability (17,33). Certain SH2 mutations give host-dependent phenotypes (7,8,42), indicating that the SH2 region may interact with host cell proteins. Recently, SH2 sequences have been shown to bind to phosphotyrosine-containing proteins (31).SH3 sequences are present in most proteins containing SH2, as well as in a-spectrin (28), Acanthamoeba myosin-IB (34), and the actin binding protein of Saccharomyces cerevisiae, ABPlp (9). The latter three proteins are all associated with the membrane cytoskeleton, suggesting that the SH3 sequence mediat...

The actin filament-associated protein AFAP-110 is an adaptor protein that modulates changes in actin filament integrity

“…These data indicated that perhaps AFAP-110 functioned by facilitating interactions between Src and actin ®laments (Pawson, 1995). Previous data predicted that transformation-competent forms of Src fractionated with the detergent-resistant cytoskeletal fraction, indicating that activated Src is closely associated with actin ®laments (Fukui et al, 1991). Thus, AFAP-110 is positioned to facilitate this interaction and perhaps Src/AFAP-110 interactions may be important for a ecting actin ®lament integrity in Src-transformed cells.…”

UCS15A, a non-kinase inhibitor of Src signal transduction

“…Localization of src to the membrane cytoskeleton, especially at focal adhesions, has been shown to be kinase-independent, but shows an absolute requirement for the SH3 domain of src (Kaplan et al, 1994). However, other studies point to the importance of the SH2 and catalytic domains, and not the SH3 domain, in these interactions (Fukui et al, 1991;Okamura and Resh, 1994). In either case, it is clear that proteinprotein interactions play important roles in the subcellular localization of src and its involvement in signaling complexes.…”

The many faces of Src: multiple functions of a prototypical tyrosine kinase

“…This interaction has been shown to be dependent on an intact SH3 domain, arguing that the SH3 domain is a key element in localizing Src to the detergent insoluble membrane associated fraction. However, the kinase domain itself may also play a role in some of these interactions ± mutation of the SH3 domain in the context of full-length or activated (Y527F) Src does not necessarily prevent association with the cytoskeleton, suggesting critical interactions and possible functional redundancy of localization functions with other parts of the molecule (Fukui et al, 1991;Okamura and Resh, 1994 and P Schwartzberg, unpublished observations). The exact nature of Src's localization may also depend on the pathway by which it has been activated ± activation of Src by PDGF is associated with an increase in detergentinsolubility and a translocation of Src to the plasma membrane, but not necessarily to focal adhesions.…”