High Affinity Binding of Hsp90 Is Triggered by Multiple Discrete Segments of Its Kinase Clients

Abstract

The 90 kDa heat shock protein (Hsp90) cooperates with its co-chaperone Cdc37 to provide obligatory support to numerous protein kinases involved in the regulation of cellular signal transduction pathways. In this report, crystal structures of protein kinases were used to guide the dissection of two kinases [the Src-family tyrosine kinase, Lck, and the heme-regulated eIF2alpha kinase (HRI)], and the association of Hsp90 and Cdc37 with these constructs was assessed. Hsp90 interacted with both the N-terminal (NL) and C-terminal (CL) lobes of the kinases' catalytic domains. In contrast, Cdc37 interacted only with the NL. The Hsp90 antagonist molybdate was necessary to stabilize the interactions between isolated subdomains and Hsp90 or Cdc37, but the presence of both lobes of the kinases' catalytic domain generated a stable salt-resistant chaperone-client heterocomplex. The Hsp90 co-chaperones FKBP52 and p23 interacted with the catalytic domain and the NL of Lck, whereas protein phosphatase 5 demonstrated unique modes of kinase binding. Cyp40 was a salt labile component of Hsp90 complexes formed with the full-length, catalytic domains, and N-terminal catalytic lobes of Lck and HRI. Additionally, dissections identify a specific kinase motif that triggers Hsp90's conformational switching to a high-affinity client binding state. Results indicate that the Hsp90 machine acts as a versatile chaperone that recognizes multiple regions of non-native proteins, while Cdc37 binds to a more specific kinase segment, and that concomitant recognition of multiple client segments is communicated to generate or stabilize high-affinity chaperone-client heterocomplexes.

Definition of Protein Kinase Sequence Motifs That Trigger High Affinity Binding of Hsp90 and Cdc37

“…Furthermore, the domain structure of Cdc37 has been determined, and its kinase binding and Hsp90 binding activities have been mapped to its N-terminal and middle domains, respectively (20 -22), and we have demonstrated that phosphorylation of Cdc37 at Ser 13 by casein kinase II is require for the ability of Cdc37 to bind protein kinase (23). However, although the specific basis for the recognition of protein kinases by Cdc37 is unknown, recent studies have mapped the recognition motif to the N-terminal lobe of the catalytic domain of protein kinases (12,24,25).…”

“…Analyses with these compounds, with nucleotides and nucleotide analogs, and with site direct mutants that alter Hsp90 ATP binding and/or ATPase activity have revealed that Hsp90 function is regulated via the binding and hydrolysis of ATP, which modulates the switching of Hsp90 between at least three alternative conformations (1)(2)(3)(4)32). In the presence of geldanamycin, Hsp90 binds weakly to client kinases in a salt-labile fashion (20,24,33). These aberrant Hsp90 heterocomplexes indicate that nucleotide modulation of conformational switching is required to generate high affinity interactions of Hsp90 and Cdc37 with protein kinases (20,24,33).…”

“…In the presence of geldanamycin, Hsp90 binds weakly to client kinases in a salt-labile fashion (20,24,33). These aberrant Hsp90 heterocomplexes indicate that nucleotide modulation of conformational switching is required to generate high affinity interactions of Hsp90 and Cdc37 with protein kinases (20,24,33). Like geldanamycin, the anion molybdate inhibits Hsp90 function.…”

“…Like geldanamycin, the anion molybdate inhibits Hsp90 function. However, in contrast to geldanamycin, this inhibition reflects the ability of molybdate to "lock" or freeze Hsp90 ki-nase complexes in high affinity salt-stable complexes (20,24,33). Thus, molybdate causes the accumulation of Hsp90 complexes containing the Hsp90 co-chaperones p23, Cdc37, and an assortment of other "late" complex components, such as tetratricopeptide repeat motif-containing immunophilins (20,24,(33)(34)(35)(36)(37)(38)(39).…”

“…Thus, molybdate causes the accumulation of Hsp90 complexes containing the Hsp90 co-chaperones p23, Cdc37, and an assortment of other "late" complex components, such as tetratricopeptide repeat motif-containing immunophilins (20,24,(33)(34)(35)(36)(37)(38)(39). However, although molybdate stabilizes the normally labile interaction of Hsp90 with steroid receptor clients (24,33,34), the interactions of Hsp90 with kinase clients are stable to high salt concentrations independent of molybdate freezing (20,24,33,34). Thus, we have utilized the ability of kinase constructs to trigger molybdate-independent, high affinity, salt-stable interactions with Hsp90 and Cdc37 to define motifs recognized by these chaperones (24).…”