Evidence for switch behavior in the Src family kinase, Hck

Src family protein‐tyrosine kinases (SFKs) are regulated by intramolecular binding of the SH2 domain to the C‐terminal tail and association of the SH3 domain with the SH2‐kinase linker. The presence of two regulatory interactions raises the question of whether disruption of both is required for kinase activation. To determine whether SH2‐based activation of SFKs requires SH3‐linker displacement, we engineered a high affinity linker (HAL) mutant of the Src family member Hck in which an optimal SH3 ligand was substituted for the natural linker. SPR analysis demonstrated tight intramolecular binding of the HAL sequence to SH3. Hck‐HAL was then combined with an activating tail tyrosine mutation (Y501F) and expressed in Rat‐2 fibroblasts. Surprisingly, Hck‐HAL‐Y501F showed strong transforming and kinase activities, demonstrating that intramolecular SH3‐linker release is not required for SH2‐based kinase activation or signaling. In yeast cells, which lack the negative regulatory tail kinase Csk, wild‐type Hck was more strongly activated in the presence of an SH3 binding protein (HIV‐1 Nef), suggesting that native SH3‐linker interaction persists in an active conformation of wild‐type Hck. These data complement our observation that Hck activation by SH3‐directed ligands does not require dephosphoryation of the negative regulatory tail or its release from SH2. Together, these findings suggest that Hck and other SFKs operate as OR‐gate signaling switches and adopt multiple active conformations in vivo. Research supported by NIH Grants AI57083 and GM70590.