CK2 is a serine/threonine protein kinase and an Hsp90 client [30
] that phosphorylates multiple serine and threonine residues in human Hsp90α (hHsp90α) and yeast Hsp82 (yHsp90) [31
]. Recently, we used S. cerevisiae
to show that CK2 phosphorylates a single conserved threonine residue (T22) in the N-domain of yHsp90 both in vitro
and in vivo
]. T22 resides in helix-1 of the Hsp90 N-domain that, together with other adjacent amino acids, is involved in an important hydrophobic interaction with the ATPase catalytic loop in the M-domain that helps to establish Hsp90's ATP hydrolysis-competent state.
The functional importance of T22 was uncovered initially in a genetic screen, where its mutation to isoleucine affected the chaperoning of v-Src and glucocorticoid receptor (GR) in yeast [34
]. Subsequent work demonstrated that the T22I mutant has 6-fold higher ATPase activity compared to WT yHsp90 [35
]. In contrast, we have shown that mutation of T22 to non-phosphorylatable alanine (T22A) did not affect its ATPase activity, while phospho-mimetic mutation of this residue to glutamic acid (T22E) reduced ATPase activity by 60% compared to WT yHsp90. Nevertheless, both mutants in yeast and the equivalent mutations in hHsp90α (T36A and T36E) affected Hsp90-dependent chaperoning of kinase (v-Src, Mpk1/Slt2, Raf-1, ErbB2 and CDK4) and non-kinase (cystic fibrosis transmembrane conductance regulator protein and GR) clients [33
To explore further whether ATP binding is a prerequisite for T22 phosphorylation, we examined the ability of CK2 to phosphorylate two conformationally distinct Hsp90 N-domain mutants. CK2 was able to efficiently phosphorylate yHsp90-E33A, which binds ATP equivalently to wild-type but, upon ATP binding, favors a “closed” (N-domain dimerized) conformation (i.e., is unable to hydrolyze ATP). However, CK2 was unable to phosphorylate yHsp90-D79N, which cannot bind ATP and thus favors an “open” (N-domain undimerized) conformation (Figure ). Since T22 is not accessible to solvent once ATP-dependent N-domain dimerization has occurred [18
], these data suggest that ATP binding to the open conformation of Hsp90 sets in motion rapid conformational change within and adjacent to helix-1 that is a prerequisite for CK2-mediated phosphorylation. Indeed, a recent study of the bacterial ortholog of Hsp90, HtpG, confirms that this region of the N-domain undergoes very rapid conformational change upon ATP binding that significantly precedes ATP-induced N-domain dimerization [35
] Since T22 phosphorylation slows the rate of ATP hydrolysis without affecting ATP binding, it is possible that eukaryotic cells utilize this post-translational modification to adjust the rate of the Hsp90 cycle to meet the optimal chaperone requirements of individual client proteins.
Figure 2 A) CK2-mediated threonine phosphorylation of the N-domain of WT, T22A, E33A and D79N yHsp90-His6 in vitro. Threonine phosphorylation was detected with a pan anti-phosphothreonine antibody. For more details, please see . B) WT, T22A, and T22E yHsp90-His (more ...)
We showed previously that phosphorylation of Y24 is a signal for Hsp90 polyubiquitination and degradation by cytoplasmic proteasomes [33
]. We explored the possibility that T22 phosphorylation of yHsp90 may serve a similar purpose. Yeast expressing WT Hsp90 as well as the phospho mutants (T22A and T22E) were treated with the proteasome inhibitor MG132 (50 μM for 1h). This resulted in equivalent accumulation of polyubiquitinated yHsp90 in each case (Figure ). Therefore, unlike Y24, T22 phosphorylation is not likely to be a determinant of Hsp90 degradation. Instead, one can speculate that T22 phosphorylation occurs dynamically to allow for the fine-tuning of chaperone activity in response to environmental cues.