Hould therefore result in stable tyrosine phosphorylation of the Cas SD
Hould therefore result in stable tyrosine phosphorylation of the Cas SD if they interact. The Cas SD has two sets of potential SH2 interacting tyrosines, the N-terminal subdomain containing 4 YQXP motifs located order Vadadustat between a.a. L157 to A324 and the Cterminal subdomain containing, 9 YDXP motifs located between a.a. S325 to R516. The v-crk SH2 domain interacts with YXXP motifs upon phosphorylation, suggesting that both of these regions may be able to interact with v-crk [37]. Fusions of the kinase inactive Src kinase domain and either the full Cas SD, the 5′ region of the Cas SD (Cas(5’SD), containing 4 YQXP motifs) or the 3′ of the Cas SD (Cas(3’SD), containing 9 YDXP motifs), are not stably tyrosine phosphorylated when expressed alone in cells (data not shown and see Fig. 4b). We therefore used these fusions to determine which region of the Cas SD interacts with v-crk. Initially, we expressed the SrcKM/ Cas(SD) (entire Cas SD) in Cos-7 cells alone, or along with wild type (WT) v-crk, or v-crk SH2 or SH3 mutants and assayed for tyrosine phosphorylation and association by co-immunoprecipitation (Fig. 4a). Figure 4a demonstrates that while the chimera was not stably tyrosine phosphorylated when expressed alone, expression of the SrcKM /Cas(SD) chimera along with either WT or an SH3 domain mutant of v-crk resulted in its stable tyrosine phosphorylation and co-immunoprecipitation with v-crk (Fig. 4a, lanes 2 4). However, co-expression of the SrcKM /Cas(SD) chimera along with a SH2 mutant of v-crk demonstrated that an intact v-crk SH2 domain is PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27693494 required for association with the tyrosine phosphorylated SrcKM/ Cas(SD) (Fig. 4a, lane 3). These data suggest that the kinase inactive fusion with the Cas SD is either transiently tyrosine phosphorylated in cells and v-crk protects them from being dephosphorylated by interacting with them via its SH2 domain, or alternatively, that v-crk induces their tyrosine phosphorylation.We then used the 5′ and 3′ fusions of the Cas SD to determine which motif, YQXP or YDXP, interacts with the v-crk SH2 domain. Expression of the SrcKM fused to the full length Cas SD, 5′ N-terminal YQXP motifs or 3′ C-terminal YDXP motifs alone demonstrated that these fusions were not stably tyrosine phosphorylated in cells (Fig. 4b, lanes 1?), although they can be stably tyrosine phosphorylated when fused to the attenuated Src kinase domain (data not shown and see Fig. 7a). By contrast, co-expression of these fusions along with v-crk results in stable tyrosine phosphorylation of the 3′ C-terminal YDXP motifsPage 3 of(page number not for citation purposes)BMC Cell Biology 2002,http://www.biomedcentral.com/1471-2121/3/Figure 2 Structure of attenuated Src kinase domain/Cas chimeras. The Src/Cas(SD) chimeras were constructed as described in the materials and methods. Src* is the isolated attenuated Src kinase domain (Y416F). Src*/Cas(SD) is a fusion of the attenuated Src kinase domain to a.a. L157 to R516 of the Cas substrate domain. Src*/Cas(5’SD) is a fusion of the attenuated Src kinase domain to the 5′ portion of the Cas substrate domain (a.a. L157 to A324). Src*/Cas(3’SD) is a fusion of the attenuated Src kinase domain to the 3′ portion of the Cas substrate domain (a.a. S325 to R516). SrcKM is the isolated inactive kinase domain (K295M). SrcKM/Cas(5’SD) is a fusion of the inactive Src kinase domain to the 5′ portion of the Cas substrate domain (a.a. L157 to R516). SrcKM/Cas(3’SD) is a fusion of the inactive Src kinase domain to the.
