There is accumulating evidence that Cdc37 has a general role in kinase biogenesis, but there has been no systematic analysis at a kinomic level. In our studies, we addressed this issue by analysis of ~50% of the yeast kinome for dependence on Cdc37. Our results are consistent with Cdc37 having a general role in kinome biogenesis and functioning either during or immediately after translation to protect nascent chains from degradation. In addition, Cdc37 promotes kinase maturation to the folded state.
The finding that Cdc37 plays a general role in kinase biogenesis is consistent with the growing number of kinases that have been characterized to interact with this chaperone (MacLean and Picard, 2003
). Two other large-scale screens for Hsp90-interacting proteins also uncovered protein kinases. In a two-hybrid screen with a mutant form of Hsp90 as bait (Millson et al., 2005
), six kinases were identified as interacting with Hsp90, and, in a multiapproach study (Zhao et al., 2005
), 27 kinases interacted with Hsp90 either physically or genetically. Interestingly, the kinases found in these studies did not overlap, indicating that neither screen was saturating.
The results of our studies show that Cdc37 functions immediately after translation to protect nascent kinase chains from degradation. Even after a subminute pulse labeling, we noted that kinase levels were reduced in the cdc37S14A mutant strain (unpublished data). However, proteasome inhibition followed by pulse labeling largely restored Tpk2, Pho85, and Rim11 levels, providing strong support for Cdc37's role in nascent chain quality control. This phenotype was only observed when MG132 was incubated for brief periods (30 min) in the cells before pulse labeling. Prolonged treatments (2 h) failed to restore Tpk2 levels, although this may result from reduced transcription occurring from long-term proteasome inhibition (unpublished data).
The finding that some kinases require Cdc37 to protect them against rapid degradation shortly after synthesis suggests that the chaperone functions as a conformational sensor at or near to the polysome. The degradation of several kinases during pulse labeling is so fast that we initially considered that translation itself was impaired. However, polysome profiles and measurement of the rate of protein synthesis suggested that Cdc37 was not involved in regulating translation, at least in a general manner (Fig. S2). Also, there is a precedent for the very rapid degradation of ribosomal proteins shortly after synthesis (Warner et al., 1985
; Maicas et al., 1988
). Although it is unclear whether this quality control pathway is the same as the one observed here, a recent study has discovered a role for Hsp90 in the biogenesis of some ribosomal proteins (Maicas et al., 1988
; Kim et al., 2006
Therefore, our results are consistent with the hypothesis that Cdc37 protects some nascent kinase chains from rapid degradation immediately after translation. Whether Cdc37 binds directly to the nascent chain as it is being synthesized or binds immediately afterward may depend on where in the polypeptide chain the kinase domain is located. Scroggins et al. (2003)
have observed Cdc37 binding to polysomes programmed with heme-regulated inhibitor kinase, for example, but not with lymphocyte-specific protein tyrosine kinase. In our own studies, we failed to observe Cdc37 binding to polysomes isolated from yeast even when such polysomes were enriched for Ste11 kinase mRNA (unpublished data). What remains unclear is how such a function for Cdc37 is integrated with those of Hsp70/40 chaperones that interact with misfolded kinases before Cdc37 binding, at least in vitro (Arlander et al., 2006
). The function of Cdc37 in stabilizing kinase nascent chains suggests that it belongs to the newly defined group of chaperones linked to protein synthesis (Albanese et al., 2006
). This group includes others that are known to promote protein kinase biogenesis such as Ydj1 and Sse1. However, Cdc37 appears to be distinct from this group in other ways because the cdc37
mutant did not display hypersensitivity to a translation inhibitor nor is CDC37
transcriptionally coregulated with other components of the translational machinery. As pointed out by Albanese et al. (2006)
, however, some chaperones appear to function as both chaperones linked to protein synthesis and as stress-regulated chaperones that function in protein refolding, and this appears to apply to Cdc37.
Rapid degradation of nascent kinase chains in the cdc37
mutant during pulse labeling was not observed in all cases. Cdc28 levels were similar in wild-type and cdc37
mutant strains after pulse labeling, but the kinase was degraded rapidly within a 30-min period. These findings are consistent with previous studies showing that Cdc37 promotes Cdc28 stability (Gerber et al., 1995
; Farrell and Morgan, 2000
). Whether Cdc28 represents a distinct class of kinase that is degraded by a different pathway is unclear. In our experiments, Cdc28 was untagged, contrasting with the C-terminal TAP-tagged kinases used in all other experiments and providing a possible source for the phenotypic distinction. However, it is also possible that Cdc28 is protected in its prefolded form by other chaperones and that loss of Cdc37 function manifests at a slightly later stage in its maturation. This explanation for the delayed degradation of Cdc28 compared with Tpk2 or Rim11 may also account for why geldanamycin promotes degradation only after kinase synthesis. As shown in , Tpk2 is completely synthesized and stable for the duration of a 5-min pulse even in the presence of geldanamycin, although it is rapidly degraded thereafter. These results are consistent with the posttranslational role attributed to Hsp90 and with nascent kinases being stabilized by other chaperones, including Cdc37 and Ydj1/Hsp70, before interacting with Hsp90 for the final stages in maturation.
The temperature-sensitive phenotype for kinase stability in the cdc37S14A
mutant allowed us to further dissect the different roles for this chaperone in kinase biogenesis. For Rim11 and Tpk2, the increase in kinase stability in cdc37S14A
when grown at 26°C did not lead to full activity; rather, a twofold decrease in activity was observed. For Tpk2, the decrease in activity of the isolated kinase correlated with decreased binding of its inhibitory subunit Bcy1 in cell lysates. This could be caused by Tpk2 misfolding and/or by increased cAMP levels in the cdc37S14A
cells caused by a combined decrease in Tpk1/2/3 activity (Nikawa et al., 1987
). These combined results are consistent with decreased kinase activity in cdc37S14A
mutant cells even when the triage system for degrading misfolded kinases is suppressed by growth at low temperature.
A previous study supports the hypothesis that kinases can accumulate as misfolded conformers that are not degraded. In this case, the deletion of STI1
resulted in very low v-Src levels, an effect that was suppressed by the overexpression of full-length or truncated forms of Cdc37 (Lee et al., 2002
). Although the full-length Cdc37 also restored v-Src activity, the truncated forms promoted stabilization without large increases in activity. Because v-Src is constitutively active in the absence of other kinases, these data indicate that v-Src accumulates in an improperly folded form that is not degraded. The same is likely true for Sky1, which is also constitutively active (Nolen et al., 2001
). Furthermore, the accumulation of misfolded proteins upon the deletion of several different chaperone proteins was recently demonstrated (McClellan et al., 2005
). In this case, components of the Hsp90 chaperone machinery were implicated in promoting degradation but not in the folding of a heterologously expressed protein in yeast. For kinases, the triage system may operate to clear the cell of misfolded kinases close to their site of synthesis before they have a chance to aggregate. This can be bypassed by growth at low temperature even in the absence of chaperone function (). In this case, the effect of low temperature may compensate for decreased chaperone function and allow the kinases to proceed to a later stage of maturation with a greater probability of achieving the native state. We suspect this has physiological relevance because some kinases interact persistently with chaperones even after initial folding. For these kinases, chaperones are constantly needed to promote the folded state rather than target the polypeptide for degradation.
In conclusion, our findings show that Cdc37 has a general role in protecting nascent kinase chains from degradation in addition to its function in posttranslational maturation. These findings suggest that Cdc37 is a gatekeeper to cellular kinase abundance. Importantly, yeast Cdc37 synthesis is fairly constant compared with other chaperones whose expression is stress regulated, and this may be a means of limiting kinase abundance in cells.