Hsp90 Regulates p50 Function during the Biogenesis of the Active Conformation of the Heme-regulated eIF2α Kinase

Abstract

Recent studies indicate that p50cdc37 facilitates Hsp90-mediated biogenesis of certain protein kinases. In this report, we examined whether p50 cdc37 is required for the biogenesis of the heme-regulated eIF2␣ kinase (HRI) in reticulocyte lysate. p50 cdc37 interacted with nascent HRI co-translationally and this interaction persisted during the maturation and activation of HRI. p50 cdc37 stimulated HRI's activation in response to heme deficiency, but did not activate HRI per se. p50 cdc37 function was specific to immature and inactive forms of the kinase. Analysis of mutant Cdc37 gene products indicated that the N-terminal portion of p50 cdc37 interacted with immature HRI, but not with Hsp90, while the C-terminal portion of p50 cdc37 interacted with Hsp90. The Hsp90-specific inhibitor geldanamycin disrupted the ability of both Hsp90 and p50 cdc37 to bind HRI and promote its activation, but did not disrupt the native association of p50 cdc37 with Hsp90. A C-terminal truncated mutant of p50 cdc37 inhibited HRI's activation, prevented the interaction of Hsp90 with HRI, and bound to HRI irrespective of geldanamycin treatment. Additionally, native complexes of HRI with p50 cdc37 were detected in cultured K562 erythroleukemia cells. These results suggest that p50 cdc37 provides an activity essential to HRI biogenesis via a process regulated by nucleotide-mediated conformational switching of its partner Hsp90.The heme-regulated inhibitor (HRI) 1 of protein synthesis is a protein-serine kinase which coordinates the synthesis of globin chains with the availability of heme in reticulocytes (reviewed in Refs. 1 and 2). Under heme-deficient conditions, HRI phosphorylates the ␣-subunit of eukaryotic translational initiation factor eIF2. Phosphorylation of eIF2␣ causes an inhibition of polypeptide chain initiation and the arrest of protein synthesis, preventing the synthesis of apo-globin chains in the absence of heme. HRI is also activated under heme-replete conditions in response to a host of other adverse environmental stimuli, such as heat shock, agents that generate oxidative stress, and the presence of denatured proteins (1, 2).The biogenesis and activation of HRI into an active hemeregulatable eIF2␣ kinase requires its functional interaction with the chaperone machinery containing the 90-kDa heat shock protein (Hsp90) and the 70-kDa heat shock cognate protein (Hsc70) (3, 4). During HRI biogenesis and its subsequent transformation and activation, several discrete HRI intermediates are generated; these intermediates can be distinguished on the basis of their competence to become an active kinase in response to heme deficiency or upon treatment with sulfhydryl reactive compounds such as N-ethylmaleimide. Immediately after their synthesis, HRI molecules are not active in hemereplete or heme-deficient rabbit reticulocyte lysate (RRL) and cannot be activated by N-ethylmaleimide treatment. This immature population interacts with Hsp90 and Hsc70 (3-7). Subsequent to this immature phase, a "mature-competent" HRI population appear...

Heme: a versatile signaling molecule controlling the activities of diverse regulators ranging from transcription factors to MAP kinases

“…The molecular chaperones Hsp90 and Hsc70 are critical for HRI maturation and heme regulation (Table 1) [54,[64][65][66][67][68]. Nascent HRI is not active during heme deficiency or heme sufficiency in reticulocytes [69].…”

“…However, this mature competent HRI continues to interact with the chaperones [54,69,71]. The interaction between HRI and the chaperones is thought to be important for maintaining HRI in a mature state that is capable of activation under heme deficiency [67,69]. Upon activation in heme-deficient conditions, HRI gains autokinase activity and eIF2a kinase activity [54,69,71].…”

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

“…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).…”

“…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).…”