Conformational processing of oncogenic v-Src kinase by the molecular chaperone Hsp90

Hsp90 is a molecular chaperone involved in the activation of numerous client proteins, including many kinases. The most stringent kinase client is the oncogenic kinase v-Src. To elucidate how Hsp90 chaperones kinases, we reconstituted v-Src kinase chaperoning in vitro and show that its activation is ATP-dependent, with the cochaperone Cdc37 increasing the efficiency. Consistent with in vivo results, we find that Hsp90 does not influence the almost identical c-Src kinase. To explain these findings, we designed Src kinase chimeras that gradually transform c-Src into v-Src and show that their Hsp90 dependence correlates with compactness and folding cooperativity. Molecular dynamics simulations and hydrogen/deuterium exchange of Hsp90-dependent Src kinase variants further reveal increased transitions between inactive and active states and exposure of specific kinase regions. Thus, Hsp90 shifts an ensemble of conformations of v-Src toward high activity states that would otherwise be metastable and poorly populated.The 90-kDa heat shock protein (Hsp90) is an abundant chaperone in the cytosol of eukaryotes (1). Together with its cochaperones, it functions in the conformational control of many regulatory proteins (2–4). Kinases constitute the largest group of Hsp90 client proteins with more than 60% of the human kinases that depend on Hsp90 in terms of their activity (5, 6).Hsp90 forms V-shaped homodimers connected via a C-terminal domain. The middle domain (M-domain) is involved in client binding (7, 8), and the N-terminal domain binds ATP. Upon ATP binding, the N-terminal domains dimerize, leading to the closed state (9–13), whereas the open state is regained upon ATP-hydrolysis (14). Both conformation and ATPase activity are affected by interaction with a cohort of cochaperones (15). Given the large number and diversity of client proteins, cochaperones are believed to deliver specificity in this context.The Hsp90-mediated maturation of kinases is strictly dependent on the cochaperone Cdc37 (cell division control protein 37) (16, 17) and phosphorylation of this cofactor is important for its function (18, 19). Binding of Cdc37 to Hsp90 causes inhibition of the ATPase activity of Hsp90 and has therefore been proposed to facilitate client kinase loading onto the Hsp90 machinery (20).The viral Src kinase (v-Src) is one of the most stringent known Hsp90 clients (5, 21). v-Src belongs to the family of nonreceptor tyrosine kinases, which play important roles in many cellular pathways. v-Src kinase is constitutively active and leads to the formation of sarcomas in chicken (22). It shows 98% sequence identity with its cellular counterpart c-Src (cellular Src kinase), the first identified protooncogene (23). Hsp90 binds to and stabilizes c-Src in its nascent state, but it dissociates after the kinase folding is achieved (24). Due to this complete loss of interaction, c-Src has been defined as a nonclient (5). Src consists of a unique domain followed by the SH3 and SH2 domains and a flexible linker, which connects the SH2 domain with the highly conserved kinase domain. c-Src contains an additional stretch at its C terminus that includes a tyrosine at the position 527, whose phosphorylation status regulates kinase activity (25, 26). In addition, v-Src differs from c-Src by several point mutations (27–30). Some of these were shown to increase c-Src activity in vivo and have been linked to cancer progression and metastasis in humans (31–33). Due to these differences, v-Src cannot be down-regulated and is permanently active, even in the absence of activating stimuli (26, 30). The analysis of proteins nearly identical in sequence but highly different in chaperone dependence offers an excellent model system for understanding the features that render a protein Hsp90-dependent. We used these kinases to reconstitute and dissect the chaperoning effect of Hsp90 on v-Src kinase in vitro. The analysis of chimeras comprising elements of c-Src and v-Src allowed us to determine the molecular basis of the stringent Hsp90 dependence of v-Src.