The SH2 and SH3 domains of pp60src direct stable association with tyrosine phosphorylated proteins p130 and p110.

Abstract

Transformation of chicken embryo cells with the tyrosine kinase oncogene src results in the tyrosine phosphorylation of numerous cellular proteins. We have recently generated monoclonal antibodies to individual tyrosine phosphorylated cellular src substrates, several of which are directed to the phosphotyrosine‐containing proteins p130 and p110. These proteins form stable complexes with activated variants of pp60src. Mutagenesis of the src homology domains (SH2 and SH3) of activated pp60src resulted in src variants with altered association with p130 and p110. Analysis of these variants showed that the SH3 domain was required for association of p110, while the SH2 domain contained residues necessary for the formation of the ternary complex involving p130, p110 and pp60src. Both the tyrosine phosphorylation status and pp60src association of p130 and p110 appeared to correlate, in part, with the extent of cell transformation. Biochemical analysis demonstrated that p130 and p110 were substrates of both serine/threonine and tyrosine kinases. In addition, p130 was redistributed from the nucleus to cellular membranes upon src transformation, whereas p110, which normally colocalized with cytoskeletal elements, was observed in adhesion plaques (podosomes) in src transformed cells. These data indicate that tyrosine phosphorylation of two different phosphoproteins may play a role during src transformation either by directing their interaction with pp60src, by redirecting subcellular distribution or both.

Allelic deletion at 11q23.3-q25 is an early event in cervical neoplasia

“…This protein is likely to be Cas (p130 cas ), an SH3-containing signaling molecule with a cluster of SH2-binding motifs . Cas is known to be highly phosphorylated on tyrosine residues in cells transformed by v-Crk and v-Src Kanner et al, 1991). Cas forms a stable complex with v-Crk and c-Crk in a phosphorylation-dependent manner and is a substrate for Src-family kinases (Vuori et al, 1996).…”

Regulation of mRNA splicing and transport by the tyrosine kinase activity of src

“…By contrast, the transport of immature transcripts, which leads to their accumulation in the cytoplasm, requires the presence of an activating mutation and is sensitive to the SH2 domain, indicating more speci®c requirement on the activity of the src gene. Similarly, Y527F/DSH2 has been reported to be poorly transforming in CEF cells (Kanner et al, 1991) in contrast with E378G/DSH2 (Seidel-Dugan et al, 1992). Therefore, the accumulation of partially spliced transcripts correlates with the transforming activity of src.…”

“…The contribution of these ligands to the e ector functions of src has not yet been fully delineated and, in particular, the role of the SH2 and SH3 domains in the establishment of the transformed phenotype is poorly understood. Indeed, they appear to be required or not, depending upon the nature of the activating mutation (Kanner et al, 1991;Seidel-Dugan et al, 1992) and/or the cellular context (Hirai and Varmus, 1990). The involvement of these domains in the regulation of the tyrosine kinase activity of src (Superti-Furga and Gon¯oni, 1997), may underly these divergent results.…”

The SH3 and SH2 domains are capable of directing specificity in protein interactions between the non-receptor tyrosine kinases cSrc and cYes

“…Stable complex formation between pp130cas and activated forms of Src are dependent primarily upon the integrity of the SH2 domain (Kanner et al, 1991;Sakai et al, 1994), although it is also capable of binding the Src SH3 domain (Nakamoto et al, 1996). Interactions between AFAP-110 and Src 527F are modulated by both the SH3 and SH2 domains, and disruption of either interaction is su cient to abrogate stable complex formation between these two proteins (Kanner et al, 1991;Guappone and Flynn, 1997;Guappone et al, 1998). Mutations in the Src SH3 domain or the SH3-binding motif of AFAP-110 also inhibit phosphorylation of AFAP-110 by Src in vivo (Kanner et al, 1991;Guappone and Flynn, 1997), indicating that SH3 domain interactions are important for presenting AFAP-110 for tyrosine-phosphorylation.…”

“…Interactions between AFAP-110 and Src 527F are modulated by both the SH3 and SH2 domains, and disruption of either interaction is su cient to abrogate stable complex formation between these two proteins (Kanner et al, 1991;Guappone and Flynn, 1997;Guappone et al, 1998). Mutations in the Src SH3 domain or the SH3-binding motif of AFAP-110 also inhibit phosphorylation of AFAP-110 by Src in vivo (Kanner et al, 1991;Guappone and Flynn, 1997), indicating that SH3 domain interactions are important for presenting AFAP-110 for tyrosine-phosphorylation. vYes is unable to induce high levels of phosphorylation of either AFAP-110 or pp130cas proteins in vivo (Kanner et al, 1990), thus one possible explanation of this data is that vYes is unable to form su ciently high a nity SH3 and/or SH2 domain interactions with AFAP-110 and pp130cas to allow e cient substrate presentation in vivo.…”