The specific activities of many protein kinases are controlled by various mechanisms such as activating phosphorylation and association with regulatory subunits. In addition, the amounts of protein kinases in cells should be strictly controlled by regulating the balance between their synthesis and degradation. The intimate collaboration between Cdc37 and Hsp90 in the quality and quantity control of protein kinases has been revealed, both genetically and biochemically (9
). Initially Cdc37 was simply considered a kinase-targeting subunit for Hsp90; however, recent advances disclosed that the relationship among Cdc37, Hsp90, and kinases is more complicated than was previously believed (27
). In this study, we have clearly shown that phosphorylation by CK2 regulates the function of Cdc37. The results suggest that Cdc37 is not a static bridge or glue but is rather a dynamic functional component of protein kinase complexes.
We observed that CK2-dependent phosphorylation of Cdc37 regulates the protein kinase-binding activity, but not the Hsp90-binding activity of Cdc37. We identified the phosphorylation site as Ser13 in the N-terminal region of Cdc37 both in vivo and in vitro. Previously, it was proposed that the N-terminal half of Cdc37 is responsible for the kinase binding, while the middle region of Cdc37 is required for the Hsp90-binding activity of Cdc37 (15
). This domain mapping agrees well with our present results that the modification of Cdc37 at the N-terminal extremity affected only the protein kinase binding activity of Cdc37. The mutation in Ser13 completely abolished the phosphorylation of Cdc37 in COS7 cells (Fig. ), indicating that no other site of Cdc37 is phosphorylated in vivo. It should be interesting to see whether phosphorylation of other sites of Cdc37 by other kinases can be observed in certain types of cells or after triggering cells by certain stimulations, such as growth factor treatment. Previously, it was reported that CK2 binds and phosphorylates Hsp90 (33
). In addition, CK2 phosphorylates another Hsp90 cochaperone, FKBP52, and disrupts the association between FKBP52 and Hsp90 (31
). Most of the steroid hormone receptor complexes contain Hsp90 and FKBP52 but not Cdc37, while protein kinase complexes include Hsp90 and Cdc37 but not FKBP52. Altogether, the chaperone complexes seem to be dynamically regulated by phosphorylation and dephosphorylation.
Although the association of Cdc37 and Hsp90 with protein kinases has been well documented, a stereoscopic view of the complexes has not yet been clarified. In this report, we determined that the protein kinase domain of MOK is responsible for the binding of Cdc37. This has been reported to also be the case for Raf1 kinase (15
). In addition, the protein kinase domain of Raf1 (51
) and MOK (32
) is essential for the Hsp90 binding. Thus, Hsp90 and Cdc37 may both bind to the protein kinase domain independently. The other possibility is that the associations of Cdc37 and Hsp90 to protein kinases are mutually interdependent or cooperative; i.e., the binding of Cdc37 or Hsp90 to the protein kinase domain is a prerequisite for the efficient association of the other. In this paper, we have observed that the disruption of the protein kinase binding activity of Cdc37 by Ser13 mutations inhibited the association of Hsp90 with kinases, whereas the association between Hsp90 and Cdc37 was not affected. The simplest explanation for this finding is that Cdc37 bridges between kinases and Hsp90; however, the precise structural analysis of the complexes will be required to solve the stereoscopic relationship among Cdc37, HSP90, and kinases.
CK2 is one of the most conserved protein kinases (16
), and the CK2 phosphorylation site of Cdc37 is evolutionarily conserved in all eukaryotic species (Fig. ). Thus, the regulation of Cdc37 by CK2-mediated phosphorylation should be conserved from S. cerevisiae
to human. In fact, a yeast strain with the cdc37
allele was reported to encode a Cdc37 mutant where Ser14 of Cdc37 (corresponding to Ser13 of mammalian Cdc37) was replaced by leucine, and this mutation was reported to cause growth arrest at a nonpermissive temperature (11
). The cdc37
allele was also shown to be defective in the activation of the MAP kinase kinase kinase family member Ste11 (1
) and that of the tyrosine kinase v-Src (11
). Bandhakavi et al. reported the identification of Cdc37 as a multicopy suppressor of a temperature-sensitive allele of CK2 in yeast and analyzed their genetic interaction in detail (4
). All of these genetic observations in yeast can well be explained by our present finding showing that CK2-dependent phosphorylation of Ser13 is of conserved and physiological importance for the molecular chaperone activity of Cdc37. Shao et al. reported that Ser13 of Cdc37 is phosphorylated and the mutation of Ser13 disrupts Cdc37 recruitment to complexes between Hsp90 and heme-regulated eIF2α kinase in rabbit reticulocyte lysate (48
). Thus, in combination with our data for MOK, Raf1, Akt, Aurora-B, Cdk4, MAK, MRK, and v-Src, it is natural to consider that all of the kinases that are targets for Cdc37 should be regulated by CK2-dependent phosphorylation of Cdc37 on Ser13 in various types of cells.
Many cellular functions modulated by CK2 have been described (2
). Our results here identify a regulatory mechanism of molecular chaperone Cdc37 by CK2. This implicates a novel type of expanding signaling in which multiple kinases are regulated indirectly by CK2 via Cdc37 (Fig. ). The number of signaling protein kinases and other functional molecules that are dependent on Cdc37 has been increasing (27
). Thus, we suggest an intriguing possibility that CK2 influences pleiotropic cellular functions by modulating numerous cellular Cdc37-dependent protein kinases by phosphorylating Ser13 of Cdc37. The enhanced activity of CK2 induces increased phosphorylation of Cdc37, which is essential for the chaperone activity of Cdc37 toward a variety of targets, leading to higher stabilities and activities of many protein kinases whose functions are dependent on functional Cdc37. Consequently, Cdc37 target kinases can be fully activatable by appropriate specific activators. This may be a reason why CK2 is apparently involved in many cellular signaling systems. A large number of cellular substrates that are phosphorylated by CK2 have been reported (29
), and substrates other than Cdc37 should also be involved in the pleiotropy of physiological roles of CK2.
It is notable that the overexpression of Cdc37 (52
) or CK2 (56
) can be oncogenic in mice. As the list of Cdc37-dependent proteins includes many tumorigenic kinases, such as Src, Raf1, and Akt, higher activity of CK2 would indirectly support the neoplastic activity of these protein kinases via Cdc37. The inhibition of multiple Hsp90-dependent kinases by a specific inhibitor, geldanamycin, has been demonstrated to be effective for anticancer chemotherapy (28
). Thus, direct inhibition of Cdc37, or inhibition of Cdc37 indirectly by inactivating CK2, can be a possible way to suppress multiple oncogenic target kinases simultaneously to achieve an anticancer effect.