and the relationship to Y416 phosphorylation, we examined the RS 1 site phosphorylation status of Y705 on STAT3, which is a downstream target of c-Src. For this experiment, protein loads on Western Blots were standardized to endogenous STAT3 levels using STAT3 immunoreactivity. Blots were reprobed with an antibody for phosphoY705 on STAT3, which revealed a greater level of reactivity for the open conformation of c-Src, consistent with open c-Src mutant displaying greater kinase activity. The closed conformations of cSrc conferred only trace levels of STAT3 Y705 phosphorylation, relative to the open conformation, and the wild-type was intermediate for both the Emerald-tagged and untagged c-Src proteins. These findings suggest that c-Src Y416 and STAT3 Y705 phosphorylation loosely correlate with each other, but that there is also a high basal Y416 phosphorylation when STAT3 phosphorylation is largely absent. We next investigated whether the Y416 phosphorylation patterns 
were governed by the minimal unit of c-Src for regulating activity: the SH2, SH3, kinase, and C-tail domains. For this experiment, we deleted the intrinsically disordered N-terminal region, which includes the unique region and a myristoylation sequence that selectively targets c-Src to the membrane, receptors and binding partners. cSrc-Emerald localized primarily in the cytosol, whereas the full length counterpart localized more extensively to the edge of the 8540743 cell and internal membranous structures, consistent with its previously reported membrane/endosomal localization patterns. Despite the altered cellular localization, c-Src in wild-type, open and closed forms conferred similar patterns of STAT3 Y705 phosphorylation to that of the full length protein. Y416 phosphorylation was also still abundant in the closed form of c-Src. Collectively, these results indicated the core functional unit of the SH2, SH3 and kinase domains of c-Src is sufficient to phosphorylate targets, such as STAT3, and itself by autophosphorylation of Y416. Furthermore, Y416 autophosphorylation persisted when these core domains were locked in the closed conformation. To confirm whether the Y416 phosphorylation observed in the closed conformation was dependent on autophosphorylation or occurred from endogenous kinases, we investigated the basal Y461 levels of phosphorylation in a catalytically inactive mutant of c-Src . The 18000030 K295 M mutant abolished Y416 and STAT3 Y705 phosphorylation, suggesting that endogenous proteins do not lead to the phosphorylation of Y416 and that Y416 phosphorylation arises directly through autophosphorylation. We next examined the dependency of Y416 phosphorylation on expression level of c-Src. Various doses of constitutively open and closed c-Src variants were transfected into AD293 cells with DNA titrated against a plasmid expressing a non-fluorescent Y66L GFP derivative to maintain a constant total load of DNA in the transfection. Western blots showed a dose dependent increase in STAT3 Y705 phosphorylation for constitutively open c-Src, whereas there was no phosphorylation for closed c-Src. Despite the lack of STAT3 phosphorylation for closed c-Src, there was a pronounced level of baseline Y416 phosphorylation especially at high levels of expression which suggests autophosphorylation can be decoupled from substrate phosphorylation Y416 Phosphorylation in Closed c-Src . Open c-Src displayed a proportionally far higher level of STAT3 and autophosphorylation than closed c-Src. It is also
