ition, we observed an appreciable level of basal Y416 phosphorylation in the closed repressed conformation of c-Src. Under these conditions, c-Src did not lead to STAT3 phosphorylation, suggesting that kinase activity for autophosphorylation can be decoupled from substrate phosphorylation. Our findings are consistent with previous reports that a constitutively closed YEEI mutant of the Src-family member Hck could undergo autophosphorylation. Prior studies have suggested that Src family kinases have a more complex multi-state conformational regulation than a Y-27632 dihydrochloride web two-state “open”and “closed”. Specifically, it was reported that the constitutively closed YEEI Hck mutant could be activated by the high-affinity SH3 ligand Nef, which suggests an intermediate activation state between fully closed and fully open. In 26367539 addition, further conformational states are implicated by studies showing that displacement of the SH2 domain of Hck in the high affinity linker mutant can lead to activation without SH3 displacement. Therefore, it remains plausible that the basal c-Src autophosphorylation arises due an intermediate active conformation of c-Src that still has the C-tail locked to the SH2 domain. Because STAT3 is recruited in an SH3-dependent manner to c-Src, an intermediate SH3-displaced state could result in STAT3 phosphorylation by c-Src that still has the C-tail locked to the SH2. However, our results demonstrate that these conformations are not sufficiently populated to enable efficient STAT3 phosphorylation. We propose that c-Src autophosphorylation may occur partly by the dimerization of c-Src, which we showed occurs for both open and closed c-Src, but which trends to higher amounts for the open state and at higher levels of expression. Hence, both open and closed c-Src participate in monomer-dimer equilibria that seems to influence Y416 autophosphorylation. The autophosphorylation of Y416 in the closed state in a concentration-dependent manner has intriguing implications for the mechanisms of c-Src regulation. These findings point to a model whereby Y416 autophosphorylation could act as a mechanism to prime locally concentrated closed c-Src for high activity upon kinase opening by secondary events. This mechanism of control of c-Src activity is conceivably important for rapidly modulating and amplifying c-Src activity at focal adhesions, where c-Src is locally enriched and engaged with focal adhesion kinase, which binds to c-Src through the SH2 domain in the open conformation. This process is also likely to not require membrane-anchored self-association, since we observed substantial autophosphorylation of c-Src in the closed state lacking the membrane anchoring myristoylation sequence. However the lipid anchor would be important for localized differences in 7 Y416 Phosphorylation in Closed c-Src activity where different ligands would also be co-enriched. Also, recent data for Src family member Lck suggests clustering on the cell surface is greater for the open conformation than the closed upon T cell receptor signaling, which indicates that lipid anchoring plays important roles in coordinating the clustering processes. The second implication is that because STAT3 phosphorylation is much more dependent on c-Src being in the open conformation than autophosphorylation, then priming of Y416 will not be sufficient to activate 12176911 substrate phosphorylation. Previous studies have suggested that STAT3 phosphorylation requires c- Src to have a functional SH3