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. 2004 May;24(9):4065-74.
doi: 10.1128/MCB.24.9.4065-4074.2004.

CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37

Yoshihiko Miyata  1 , Eisuke Nishida
Affiliations

CK2 controls multiple protein kinases by phosphorylating a kinase-targeting molecular chaperone, Cdc37

Yoshihiko Miyata et al. Mol Cell Biol. 2004 May.

Abstract

Cdc37 is a kinase-associated molecular chaperone whose function in concert with Hsp90 is essential for many signaling protein kinases. Here, we report that mammalian Cdc37 is a pivotal substrate of CK2 (casein kinase II). Purified Cdc37 was phosphorylated in vitro on a conserved serine residue, Ser13, by CK2. Moreover, Ser13 was the unique phosphorylation site of Cdc37 in vivo. Crucially, the CK2 phosphorylation of Cdc37 on Ser13 was essential for the optimal binding activity of Cdc37 toward various kinases examined, including Raf1, Akt, Aurora-B, Cdk4, Src, MOK, MAK, and MRK. In addition, nonphosphorylatable mutants of Cdc37 significantly suppressed the association of Hsp90 with protein kinases, while the Hsp90-binding activity of the mutants was unchanged. The treatment of cells with a specific CK2 inhibitor suppressed the phosphorylation of Cdc37 in vivo and reduced the levels of Cdc37 target kinases. These results unveil a regulatory mechanism of Cdc37, identify a novel molecular link between CK2 and many crucial protein kinases via Cdc37, and reveal the molecular basis for the ability of CK2 to regulate pleiotropic cellular functions.

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Figures

FIG. 1.
FIG. 1.
Phosphorylation of Cdc37 by CK2 in vitro. Purified recombinant rat Cdc37 was incubated with or without purified CK2 in the presence of radiolabeled ATP. (A and B) The phosphorylation mixtures were analyzed by SDS-PAGE followed by CBB staining (A) or by autoradiography (B). Lane 1, CK2; lane 2, Cdc37; lane 3, CK2 and Cdc37. (C and D) Phosphorylation of various deletion mutants of Cdc37 by CK2 in vitro. Various deletion mutants of recombinant Cdc37 were incubated with CK2 in the presence of radiolabeled ATP in vitro. The phosphorylation mixtures were analyzed by SDS-PAGE followed by CBB staining (C) or autoradiography (D). Deletion mutants used were Cdc37(N093) (lane 1) Cdc37(N189) (lane 2), Cdc37(N284) (lane 3), Cdc37(WT) (lane 4), Cdc37(D105) (lane 5), Cdc37(D206) (lane 6), and Cdc37(D275) (lane 7). (E) The structures of Cdc37 deletion mutants and their phosphorylation by CK2 are schematically illustrated with numbers of amino acids. +, phosphorylated by CK2; −, not phosphorylated by CK2.
FIG. 2.
FIG. 2.
Mutation in conserved Ser13 of Cdc37 abolished the phosphorylation of Cdc37 by CK2. (A) The alignment of amino acid sequences from N-terminal regions of Cdc37 for various species is shown. The positions of three serines and two threonines are shown by shaded boxes and asterisks. A CK2 target phosphorylation site is indicated as “13” according to the amino acid number of rat Cdc37. The names of the species are shown on the left with their GenBank accession numbers in parentheses. (B and C) Effect of Ser13 mutations of Cdc37 on the CK2-mediated phosphorylation in vitro. Recombinant Cdc37 and its Ser13 mutants were incubated with CK2, and the phosphorylation mixtures were analyzed by SDS-PAGE followed by CBB staining (B) or autoradiography (C). Lane 1, Cdc37(WT); lane 2, Cdc37(13SA); lane 3, Cdc37(13SD). (D and E) Mutations on Ser13 abolished the phosphorylation of Cdc37 in vivo. The equal expression of Cdc37 and its mutants in cells was shown by Western blotting of cell extracts with anti-HA antibody (D). The incorporation of phosphate in vivo into Cdc37 was visualized by 33P autoradiography after immunoprecipitation (E). Lane 1, control (nontransfected); lane 2, Cdc37(WT); lane 3, Cdc37(13SA); lane 4, Cdc37(13SD).
FIG. 3.
FIG. 3.
Protein kinase domain of MOK was essential for the binding of Cdc37. Various MOK deletion mutants with a FLAG tag were expressed in COS7 cells and immunoprecipitated. (A) Amounts of MOK and its mutants in the immunoprecipitates were examined by Western blotting. Bands corresponding to deletion mutant proteins are indicated by asterisks [except MOK(N107) (see the text)]. Lane 1, control (not transfected); lane 2, MOK(WT); lane 3, MOK(N107); lane 4, MOK(N178); lane 5, MOK(N285); lane 6, MOK(D8-078); lane 7, MOK(D8-195); lane 8, MOK(D8-309). (B) Associations of endogenous Cdc37 with the same set of MOK deletion mutants as in panel A were examined by coimmunoprecipitation experiments and revealed by Western blotting with anti-Cdc37 antibody. Lane marks are the same as those for panel A. The positions of Cdc37, immunoglobulin heavy-chain (HC), immunoglobulin light-chain (LC), and molecular weight markers are indicated. (C) The relationship between structures and the Cdc37 binding abilities of MOK deletion mutants is schematically illustrated. ++, binds strongly; +, binds well; −, no binding.
FIG. 4.
FIG. 4.
The CK2-phosphorylated form of Cdc37 was active in protein kinase binding. (A) Association of the endogenous phosphorylated form of Cdc37 and Hsp90 with MOK in vivo. FLAG-MOK and its associated proteins were isolated by immunoprecipitation from radiolabeled cells. Lane 1, control; lane 2, immunoprecipitation with anti-FLAG antibody. The positions of Hsp90, Cdc37, and MOK are shown. (B) Disruption of the CK2 phosphorylation site of Cdc37 abolished the association of Cdc37 with MOK. FLAG-MOK was expressed in COS7 cells and immunoprecipitated with anti-FLAG antibody. The association of Cdc37 was examined by Western blotting (WB) of the immunoprecipitates. Lane 1, control; lane 2, MOK only; lane 3, MOK plus Cdc37(WT); lane 4, MOK plus Cdc37(13SA); lane 5, MOK plus Cdc37(13SD). Cell extract was included in lane 6 to show the migration position of Cdc37. (C) The equality of immunoprecipitated MOK was confirmed by Western blotting of the immunoprecipitates with anti-FLAG antibody. Lane marks are the same as those for panel B. The position of cross-reacted immunoglobulin heavy-chain bands is shown by an asterisk. The position of MOK was revealed in lane 6 by longer exposure. (D) Ser13 of Cdc37 is important for the stable association with MOK. Cdc37 or its mutants were immunoprecipitated and the association of MOK was examined by Western blotting. In lane 1, extract of cells was included to show the migration position of MOK; lane 2, MOK only; lane 3, MOK plus Cdc37(WT); lane 4, MOK plus Cdc37(13SA); lane 5, MOK plus Cdc37(13SD). Positions of MOK and faint nonspecific bands (asterisk) are indicated on the right.
FIG. 5.
FIG. 5.
Disruption of the CK2 phosphorylation site of Cdc37 abolished the association of Cdc37 with Raf1. The binding of Raf1 to Cdc37 was examined by coimmunoprecipitation experiments. (A) Raf1 was immunoprecipitated from cotransfected cell lysates, and the association of Cdc37 was revealed by anti-HA Western blotting (WB). Lane 1, cell extract; lane 2, Raf1 only; lane 3, Raf1 plus Cdc37(WT); lane 4, Raf1 plus Cdc37(SA); lane 5, Raf1 plus Cdc37(SD). (B) The equal amount of Raf1 was immunoprecipitated from cotransfected cell lysates. Lane marks are the same as those for panel A. (C) Cdc37 or its mutants were immunoprecipitated from cotransfected cell lysates. The amounts of immunoprecipitated Cdc37 were revealed by Western blotting with anti-HA antibody. Lane marks are the same as those for panel A. (D) Cdc37 or its mutants were immunoprecipitated, and the amounts of Cdc37-associated Raf1 were examined by Western blotting with anti-FLAG antibody. Lane marks are the same as those for panel A.
FIG. 6.
FIG. 6.
Phosphorylation of Ser13 of Cdc37 by CK2 is important for the Hsp90 recruitment, but not for the Hsp90 binding, of Cdc37. (A) Ser13 mutations of Cdc37 diminished the association of Hsp90 with MOK. MOK and Cdc37 were cotransfected, and MOK was immunoprecipitated. The association of endogenous Hsp90 with Cdc37-MOK complexes was examined by anti-Hsp90 Western blotting (WB). Lane 1, control; lane 2, MOK only; lane 3, MOK plus Cdc37(WT); lane 4, MOK plus Cdc37(SA); lane 5, MOK plus Cdc37(13SD); lane 6, cell extract to show the position of Hsp90. (B) Ser13 mutations of Cdc37 abolished the association of Hsp90 with Raf1. The association of Hsp90 with Cdc37-Raf1 complexes was examined by anti-Hsp90 Western blotting of Raf1 immunoprecipitates. Lane 1, cell extract; lane 2, Raf1 only; lane 3, Raf1 plus Cdc37(WT); lane 4, Raf1 plus Cdc37(13SA); lane 5, Raf1 plus Cdc37(13SD). (C) Phosphorylation on Ser13 of Cdc37 was not important for Hsp90-binding activity of Cdc37. Cdc37(WT) (lanes 3 and 7), Cdc37(13SA) (lanes 4 and 8), or Cdc37(13SD) (lanes 5 and 9) with an HA tag was expressed in COS7 cells and immunoprecipitated with control antibody (lanes 2 to 5) or with anti-HA antibody (lanes 6 to 9). Nontransfected cells were used as controls (lanes 2 and 6). The coimmunoprecipitation of Hsp90 with Cdc37 was examined by anti-Hsp90 Western blotting. Cell extract was included in lane 1 to show the migration position of Hsp90. The positions of molecular weight markers are shown on the left.
FIG. 7.
FIG. 7.
Inhibition of CK2 in vivo induced dephosphorylation of Cdc37 and destabilization of Cdc37 target kinases. (A) Suppression of Cdc37 phosphorylation in vivo by CK2 inhibition. Cells were treated with 0 (lane 1), 66 (lane 2), or 166 (lane 3) μM TBB for 16 h. The phosphorylation of Cdc37 in vivo was determined by immunoprecipitation followed by SDS-PAGE and autoradiography. (B) Inhibition of CK2 in vivo induced destabilization of Cdc37 target kinases MOK and Raf1. MOK or Raf1 was expressed in COS7 cells, and the amounts of MOK (lanes 4 to 6) and Raf1 (lanes 7 to 9) after treatment of cells with 0 (lanes 1, 4, and 7), 66 (lanes 2, 5, and 8), or 166 (lanes 3, 6, and 9) μM TBB for 16 h were determined by Western blotting (WB). (C and D) The amounts of ERK2 (C) and Cdc37 (D) were determined by Western blotting with corresponding antibodies. Lane marks are the same as those for panel B. The positions of molecular weight markers (left) and corresponding proteins (right) are shown.
FIG. 8.
FIG. 8.
The CK2 phosphorylation site of Cdc37 is essential for efficient binding to multiple signaling protein kinases. Protein kinases were expressed in COS7 cells with Cdc37(WT), Cdc37(13SA), or Cdc37(13SD). Protein kinases were immunoprecipitated, and the binding of Cdc37 was revealed by Western blotting. (A) Akt; (B) Aurora-B; (C) Cdk4; (D) MAK; (E) MRK; (F) v-Src. Lane 1, cell extract to show the position of Cdc37; lane 2, control (kinase only); lane 3, kinase plus Cdc37(WT); lane 4, kinase plus Cdc37(SA); lane 5, kinase plus Cdc37(SD). (G) A schematic illustration of the expanding signal transduction from CK2 to multiple protein kinases via Cdc37 is shown.
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