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

The c-src and c-yes proto-oncogenes encode 60 000 and 62 000 Dalton non-receptor tyrosine kinases of the Src family, pp60c-src and pp62c-yes, respectively. These kinases are over 80% homologous outside of their unique amino termini, yet several studies suggest that differences exist in the regulation, activation, and function of cSrc and cYes. The determinants of specificity in signaling between these proteins, however, remain unclear. In order to investigate the roles of the Src Homology (SH) 3 and 2 domains in mediating signaling specificity between cSrc and cYes, chimeras were created in which the SH3 and/or SH2 domains of cSrc or the fully activated variant Src527F were replaced by the corresponding domains of cYes. These constructs were used to assess the effects of the Yes SH3 and SH2 domains on the ability of Src to form stable complexes with and induce tyrosine phosphorylation of Src SH3 and SH2 domain binding partners in vivo. Both the Yes SH3 and SH2 domains were found to alter the capacity of Src to form stable associations with heterologous proteins. The Yes SH3 domain was unable to affinity absorb the Src SH3/SH2 binding partner AFAP-110 from COS-1 cell lysates, and chimeric constructs of Src527F containing the cYes SH3 domain were unable to efficiently co-immunoprecipitate with AFAP-110 from chicken embryo fibroblasts. Interactions with the Src SH2 domain binding partner pp130cas were unaffected. Additionally, only chimeras containing the cYes SH2 domain were able to co-immunoprecipitate with an unidentified 87 kDa tyrosine-phosphorylated protein. These results indicate that the SH3 and SH2 domains are capable of directing specificity in substrate binding between Src and Yes, suggesting potential mechanisms for generating specificity in signaling between these two highly related non-receptor tyrosine kinases.

Monoclonal antibodies directed against AFAP-110 recognize species-specific and conserved epitopes.

The actin filament-associated protein, AFAP-110, is a Src SH2/SH3 binding partner that can modulate changes in actin filament structure. AFAP-110 contains a carboxy terminal motif that facilitates actin filament interactions, as well as amino terminal protein binding motifs, including an SH3 binding motif, two SH2 binding motifs, and two Pleckstrin homology domains. Two monoclonal antibodies (MAbs) were developed that recognized epitopes in either the amino terminus (MAb 4C3) or the carboxy terminus (anti-AFAP-110) of AFAP-110. Site-directed mutations that change key proline residues to alanine in the SH3 binding motif and an adjacent proline-rich motif abrogated MAb 4C3 binding. These same mutations have been shown to prevent SH3 interactions between AFAP-110 and Src527F. These data indicate that MAb 4C3 recognizes an epitope that is part of the SH3 binding motif. Interestingly, MAb 4C3 is not efficiently reactive with mammalian homologs of AFAP-110. Sequence analysis of a putative cDNA clone that encodes the amino terminus of the human AFAP-110 isoform predicted a one amino acid difference within this epitope, indicating a mechanism for species-specific binding by MAb 4C3. A second, MAb anti-AFAP-110, recognizes AFAP-110 across species and binds to an epitope within the carboxy terminus. This epitope includes the 5th heptad repeat of the carboxy terminal, leucine zipper motif (amino acids 592-598)--a motif that facilitates self-associations and may regulate the function of AFAP-110. These MAbs will be useful for analyzing the effects of AFAP-110 upon cell morphology and actin filament integrity. In addition, the avian-specific MAb 4C3 may be useful for studying the effects of avian AFAP-110 constructs expressed in mammalian cells, by providing an internal epitope tag.

Vascular endothelial growth factor receptor-1 mediates migration of human colorectal carcinoma cells by activation of Src family kinases.

Vascular endothelial growth factor (VEGF) is the predominant pro-angiogenic cytokine in human malignancy, and its expression correlates with disease recurrence and poor outcomes in patients with colorectal cancer. Recently, expression of vascular endothelial growth factor receptors (VEGFRs) has been observed on tumours of epithelial origin, including those arising in the colon, but the molecular mechanisms governing potential VEGF-driven biologic functioning in these tumours are not well characterised. In this report, we investigated the role of Src family kinases (SFKs) in VEGF-mediated signalling in human colorectal carcinoma (CRC) cell lines. Vascular endothelial growth factor specifically activated SFKs in HT29 and KM12L4 CRC cell lines. Further, VEGF stimulation resulted in enhanced cellular migration, which was effectively blocked by pharmacologic inhibition of VEGFR-1 or Src kinase. Correspondingly, migration studies using siRNA clones with reduced Src expression confirmed the requirement for Src in VEGF-induced migration in these cells. Furthermore, VEGF treatment enhanced VEGFR-1/SFK complex formation and increased tyrosine phosphorylation of focal adhesion kinase, p130 cas and paxillin. Finally, we demonstrate that VEGF-induced migration is not due, at least in part, to VEGF acting as a mitogen. These results suggest that VEGFR-1 promotes migration of tumour cells through a Src-dependent pathway linked to activation of focal adhesion components that regulate this process.