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Hsp90 Promotes Kinase Evolution

Heat-shock protein 90 (Hsp90) promotes the maturation and stability of its client proteins, including many kinases. In doing so, Hsp90 may allow its clients to accumulate mutations as previously proposed by the capacitor hypothesis. If true, Hsp90 clients should show increased evolutionary rate compared with nonclients; however, other factors, such as gene expression and protein connectivity, may confound or obscure the chaperone’s putative contribution. Here, we compared the evolutionary rates of many Hsp90 clients and nonclients in the human protein kinase superfamily. We show that Hsp90 client status promotes evolutionary rate independently of, but in a small magnitude similar to that of gene expression and protein connectivity. Hsp90’s effect on kinase evolutionary rate was detected across mammals, specifically relaxing purifying selection. Hsp90 clients also showed increased nucleotide diversity and harbored more damaging variation than nonclient kinases across humans. These results are consistent with the central argument of the capacitor hypothesis that interaction with the chaperone allows its clients to harbor genetic variation. Hsp90 client status is thought to be highly dynamic with as few as one amino acid change rendering a protein dependent on the chaperone. Contrary to this expectation, we found that across protein kinase phylogeny Hsp90 client status tends to be gained, maintained, and shared among closely related kinases. We also infer that the ancestral protein kinase was not an Hsp90 client. Taken together, our results suggest that Hsp90 played an important role in shaping the kinase superfamily.

. The average strong client kinase dN/dS was always greater than the non-client kinase dN/dS across mammals, regardless of species used for pairwise dN/dS comparison (ratios were always greater than 1, red line).   the Hsp90 clients tended to show greater dN/dS than non-clients. b) The TK and TKL groups were enriched for strong Hsp90 clients; the CAMK group was most depleted for strong Hsp90 clients. The expected number of non-clients, weak clients, and strong clients was calculated by assuming an equal distribution of all client states across all kinase groups. c) The TK and TKL families show the largest median values of dN/dS, albeit their dN/dS did not significantly differ from the other kinase groups.

Figure S5. Family-specific differences for the association of Hsp90 client status and dN/dS.
In general, strong kinase clients (red) tended to have greater dN/dS than weak kinase clients (pink), and weak kinase clients (purple) tended to have greater dN/dS than kinase non-clients.
The TK and CAMK groups did not follow this pattern, with weak kinase clients tending to have greater dN/dS, presumably due the broad distribution of dN/dS for weak kinases in these groups.

Supplementary Text
Interaction with Hsp90 is associated with faster evolutionary rates in clients, but not cochaperones or otherwise collaborating proteins.
We failed to detect an effect of Hsp90 client status on the evolutionary rate of transcription factors and E3 ligases ( Figure S2). In contrast to the tested kinases, which evolved from a common ancestor, transcription factors and E3 ligases are not monophyletic (Li, et al. 2008;Vaquerizas, et al. 2009) and hence likely differ greater in other features influencing evolutionary rate. Hence, we subdivided the tested transcription factors (Vaquerizas, et al. 2009) and E3 ligases (Li, et al. 2008) into phylogenetically related groups for further tests.
For transcription factors, this subdivision approach was hampered by the extremely low number of transcription factors that interact with Hsp90 (58 out of 843 tested) (Taipale, et al. 2012).
Comparing evolutionary rates of clients and non-clients in phylogenetically related groups was not feasible (average group size 18.4, average number of clients 1.8, and non-clients 16.7).
For E3 ligases, we used previously described subdivisions and tested for association of Hsp90 client status and dN/dS. Specifically, we divided the E3 ligases into RING finger domaincontaining (RNF) and non-RNF proteins (Li, et al. 2008). We found no significant effect of Hsp90 client status on RNF and non-RNF E3 ligases. Similarly, testing only E3 ligases with a Kelch fold (Taipale, et al. 2012) yielded no evidence that Hsp90 interactors evolved faster than non-interactors. E3 ligases also contain small domains with roles in recognition of ubiquitinated substrates. We attempted to cluster the tested E3s by these domains as identified in Pfam, using several different thresholds for domain identity. Again, we found no significant differences in dN/dS associated with Hsp90 interaction in these groups.
This persistent failure in light of the clear and consistent signal in kinases prompted us to further explore the literature on Hsp90 and E3 ligase interaction. In contrast to TFs and kinases, which require Hsp90's assistance to reach their mature fold, E3 ligases may interact physically with Hsp90 for another reason. Indeed, some E3 ligases have been shown to collaborate with Hsp90 in the degradation of other proteins, rather than being chaperoned by Hsp90 (Murata et al. 2001;Giannini & Bijlmakers 2004;Morishima et al. 2008). If E3 ligases generally are Hsp90-collaborating proteins, we would not expect an Hsp90-associated effect on E3 ligase dN/dS, akin to prior observations with other Hsp90-associated proteins such as Hsp70 and various Hsp90 cochaperones, all of which are rather highly conserved across eukaryotes.