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

Abstract

The c-src and c-yes proto-oncogenes encode 60 000 and 62 000 Dalton non-receptor tyrosine kinases of the Src family, pp60 c-src and pp62 c-yes , respectively. These kinases are over 80% homologous outside of their unique amino termini, yet several studies suggest that di erences exist in the regulation, activation, and function of cSrc and cYes. The determinants of speci®city 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 speci®city between cSrc and cYes, chimeras were created in which the SH3 and/or SH2 domains of cSrc or the fully activated variant Src 527F were replaced by the corresponding domains of cYes. These constructs were used to assess the e ects 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 a nity absorb the Src SH3/SH2 binding partner AFAP-110 from COS-1 cell lysates, and chimeric constructs of Src 527F containing the cYes SH3 domain were unable to e ciently coimmunoprecipitate with AFAP-110 from chicken embryo ®broblasts. Interactions with the Src SH2 domain binding partner pp130cas were una ected. Additionally, only chimeras containing the cYes SH2 domain were able to co-immunoprecipitate with an unidenti®ed 87 kDa tyrosine-phosphorylated protein. These results indicate that the SH3 and SH2 domains are capable of directing speci®city in substrate binding between Src and Yes, suggesting potential mechanisms for generating speci®city in signaling between these two highly related non-receptor tyrosine kinases. Oncogene (2000) 19, 155 ± 160.

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

“…Cells expressing AFAP-110 and Src 527F , AFAP-110 and cSrc, of AFAP-110 alone showed a signi®cant portion of AFAP-110 in the triton-soluble fraction after 3 min which was increased after 10 min of triton extraction. Fractions are labeled as: T-total lysate, S-Triton-soluble and I-Triton-insoluble SH3 domain of cYes was substituted into Src 527F failed to form a stable complex with AFAP-110 as well (Summy et al, 2000). These data indicate that SH3 interactions are required for stable complex formation with AFAP-110 and that speci®city in signaling between cSrc and cYes might be achieved partly through di erential SH3 binding partners.…”

“…Co-expression studies demonstrated that the AFAP 71A mutant failed to form a stable complex with Src 527F indicating a role for SH3 interactions in mediating stable complex formation. In addition, a nity absorption experiments using the Lyn or Fyn SH3 domains (GST-SH3 lyn and GST-SH3 fyn ) demonstrated that these fusion proteins could also a nity absorb AFAP-110; however, the cYes SH3 domain (GST-SH3 yes ) was unable to absorb cellular AFAP-110 (Flynn et al, 1993;Guappone and Flynn, 1997;Summy et al, 2000). These data indicate a mechanism for generating speci®city in signaling among Src family kinases via SH3 interactions.…”

“…As mentioned earlier, AFAP-110 can be a nity absorbed by GST-fusion proteins encoding the Lyn and Fyn SH3 domains. In addition, GST-SH2 fusion proteins representing Lyn, Fyn and even cYes are also able to a nity absorb AFAP-110 (Flynn et al, 1993;Guappone et al, 1998;Guappone and Flynn, 1997;Summy et al, 2000). Because AFAP-110 is not an SH3 binding partner for cYes, and these interactions are important for working in concert with SH2 binding to facilitate stable complex formation with Src, then it was hypothesized that cYes would not be a binding partner for AFAP-110.…”

UCS15A, a novel small molecule, SH3 domain-mediated protein–protein interaction blocking drug

“…UCS15A very e ectively disrupted the interaction between Src and Sam68 (Figure 1) whereas it was somewhat less e ective in disrupting the Sam68 ± Grb2, Sam68 ± PLCg (Figure 3), Cortactin ± ZO1, Grb2 ± Sos1 and Grb2 ± Gab1 (Figure 4) complexes. Previous studies using degenerate peptide libraries and phage display libraries have suggested that even though the binding of SH3 domains to their ligands is generally characterized by relatively low a nity, they, nonetheless, exhibit distinct binding preferences (Yu et al, 1994;Birge et al, 1996) and that SH3 domains of highly related protein such as cSrc and c-Yes have distinct speci®cities with respect to their interacting partners (Summy et al, 2000). In this regard, the di erences in potency of UCS15A may be related to the di erences in the binding a nities between these proteins, or the preference of UCS15A for speci®c SH3-ligand complexes.…”

Crystallographic structure of the SH3 domain of the human c‐Yes tyrosine kinase: Loop flexibility and amyloid aggregation

“…c-Src, c-Yes and Fyn are the three most homologous members of the family, with sequence identities close to 70% in their SH3 domains. Although these kinases have been postulated to perform redundant functions in the cell, their implication in tumour development is different [1] and there is an increasing body of evidence for specificity in signalling, in some instances determined by interactions mediated by their SH3 domains [1][2][3].…”