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. 2007 Aug;6(8):1363-72.
doi: 10.1128/EC.00165-07. Epub 2007 Jun 15.

Alteration of the protein kinase binding domain enhances function of the Saccharomyces cerevisiae molecular chaperone Cdc37

Min Ren  1 , Arti Santhanam, Paul Lee, Avrom Caplan, Stephen Garrett
Affiliations

Alteration of the protein kinase binding domain enhances function of the Saccharomyces cerevisiae molecular chaperone Cdc37

Min Ren et al. Eukaryot Cell. 2007 Aug.

Abstract

Cdc37 is a molecular chaperone that has a general function in the biogenesis of protein kinases. We identified mutations within the putative "protein kinase binding domain" of Cdc37 that alleviate the conditional growth defect of a strain containing a temperature-sensitive allele, tpk2(Ts), of the cyclic AMP-dependent protein kinase (PKA). These dominant mutations alleviate the temperature-sensitive growth defect by elevating PKA activity, as judged by their effects on PKA-regulated processes, localization and phosphorylation of the PKA effector Msn2, as well as in vitro PKA activity. Although the tpk2(Ts) growth defect is also alleviated by Cdc37 overproduction, the CDC37 dominant mutants contain wild-type Cdc37 protein levels. In addition, Saccharomyces cerevisiae Ste11 protein kinase has an elevated physical interaction with the altered Cdc37 protein. These results implicate specific amino-terminal residues in the interaction between Cdc37 and client protein kinases and provide further genetic and biochemical support for a model in which Cdc37 functions as a molecular chaperone for protein kinases.

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Figures

FIG. 1.
FIG. 1.
PKA and Tor kinase control of the general stress response. Cytoplasmic localization of the general stress response transcription factor Msn2 is independently stimulated by the PKA active subunit Tpk2 and the Tor kinase. Binding of cAMP to the PKA negative regulatory subunit Bcy1 stimulates the release and activation of Tpk2. Phosphorylation of the Msn2 carboxyl terminus by Tpk2 inhibits nuclear import, whereas Tor kinase acts on the Msn2 amino terminus in an unknown way to stimulate efficient nuclear export. Inactivation of either kinase results in the accumulation of Msn2 in the nucleus and the coordinate expression of genes necessary for growth inhibition (YAK1), response to stress (HSP12), and glycogen accumulation (GLC3).
FIG. 2.
FIG. 2.
The tpk2(Ts) growth defect is suppressed by alterations of the amino-terminal sequence of Cdc37. (A) Suppression of the tpk2(Ts) growth defect by the CDC37-101 mutation. Strains were streaked onto minimal medium and incubated for several days. Wild-type (WT) [SGY559(pRS316)], tpk2-63(Ts) CDC37+ [SGY446(pYZ1)], tpk2-63(Ts) CDC37-101 [SGY446(pGS224)], tpk2-62(Ts) CDC37+ [SGY562 (pYZ1)], and tpk2-62(Ts) CDC37-101 [SGY562(pGS224)] strains were used. (B) Suppression of the tpk2(Ts) growth defect in the absence of Bcy1. Strain SGY398 (tpk2-63 bcy1Δ) was transformed with a low-copy-number vector or the same vector containing wild-type (CDC37+) or mutant (CDC37-101) alleles of CDC37 and incubated on minimal medium for several days at 23°C and 34°C. (C) The dominant CDC37 mutations fall within the amino terminus of Cdc37. The individual mutations are indicated by lines within the putative protein kinase interaction domain (filled box) of Cdc37.
FIG. 3.
FIG. 3.
Mutant Cdc37 restores Msn2 function, localization, and phosphorylation to a tpk2(Ts) strain. (A) A tpk2(Ts) CDC37-101 strain accumulates wild-type levels of glycogen. Strains were patched onto rich medium agar and incubated at 23°C for several days before (−) and after (+) exposure to iodine vapors. TPK2 CDC37+ [SGY559(pRS316)], tpk2-62 CDC37+ [SGY562(pYZ1)], and tpk2-62 CDC37-101 [SGY562(pGS224)] strains were used. (B) The CDC37-101 mutation blocks Msn2-GFP from accumulating in the nucleus of a tpk2-62(Ts) strain. Cells were grown at 23°C to mid-log phase before shifting to 34°C for 2 h. Fifteen minutes before harvesting, Hoechst 33342 stain was added (50-μg/ml final concentration) to visualize DNA. Strains were derivatives of tpk2-62(Ts) strain SGY562 containing the MSN2-GFP3 fusion plasmid pADH-PKI-MSN2-(576-704)-GFP3. CDC37+ (MRY43) and CDC37-101 (MRY45) strains were used. (C) The CDC37-101 mutation partially restores Msn2 phosphorylation in a tpk2-62(Ts) strain. Strains were grown at 23°C to mid-log phase and then shifted to 34°C for 2 h. Extracts were separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and then exposed sequentially to anti-phospho-CREB antibody (α-CREB) to monitor PKA-dependent phosphorylation and to anti-GFP antibody (α-GFP) to monitor Msn2 levels. TPK2 CDC37+ (MRY31), tpk2-62 CDC37+ (MRY43), and tpk2-62 CDC37-101 (MRY45) strains were used.
FIG. 4.
FIG. 4.
The CDC37 dominant mutations alter Tpk2 activity. (A) Stability of wild-type and temperature-sensitive Tpk2 proteins. Strains containing Myc-tagged wild-type Tpk2 and mutant Tpk2-63 fusion proteins were grown at 23°C and shifted to 34°C medium containing 100 mg/ml cycloheximide. Protein extracts were separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with anti-Myc antibody. Tpk2 (MRY142) and Tpk2-63 (MRY143) strains were used. (B) Dominant CDC37 mutations do not alter Tpk2 levels. Levels of the Myc-tagged temperature-sensitive Tpk2-63 fusion protein were determined after shifting derivatives of strain MRY142 from 23°C to 34°C for 3 h. Derivatives contained plasmids with either wild-type (WT) (CDC37+) (pYZ1) or mutant 106 (CDC37-106) (pGS224) alleles of CDC37. Extracts were separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with anti-Myc antibody to measure Tpk2 levels. (C) Dominant CDC37 mutations alter the activity of purified Tpk2. Tpk2 fusion proteins were immunopurified and used in an in vitro peptide kinase assay. After 30 min, peptide from each assay was separated by agarose gel electrophoresis to determine if the peptide was unphosphorylated (−) or phosphorylated (PO4). Aliquots of the immunoprecipitate were also separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with anti-Myc antibody (α-myc) to determine if similar levels of protein kinase were present in each reaction. Strains were derivatives of Tpk2-Myc (MRY143) or Tpk2-63-Myc (MRY42) strains containing plasmids with WT (CDC37+ pYZ1) or 106 (CDC37 pGS224) alleles of CDC37. (D) Dominant CDC37 mutations alter the activity of wild-type and mutant PKA. Cell extracts from the indicated strains were used in the in vitro peptide kinase assay. TPK2 CDC37+ [SGY559(pRS316)], TPK2 CDC37-106 [SGY559(pYZ2)], tpk-63(Ts) CDC37+ [SGY446(pRS316)], and tpk-63(Ts) CDC37-106 [SGY446(pYZ2)] strains were used.
FIG. 5.
FIG. 5.
Cdc37 levels and protein interactions in the dominant CDC37 mutants. (A) Protein extracts of the indicated strains were separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose, and probed with anti-Cdc37 antibody. cdc37Δ (high-copy-number [HC] CDC37+) (MRY4), cdc37Δ (low-copy-number [LC] CDC37-109) (MRY5), cdc37Δ (low-copy-number CDC37+) (MRY8), cdc37Δ (low-copy-number CDC37-101) (MRY11), and CDC37+ (low copy number) (MRY10) strains were used. (B) Interaction of Ste11 with wild-type and mutant Cdc37. Whole-cell extracts were incubated with Ni-nitrilotriacetic acid to purify His6-tagged Ste11 proteins and separated by polyacrylamide gel electrophoresis before sequentially Western blotting with antisera to Ste11, Hsp90, and Cdc37. Extracts were made from CDC37+ (MRY73), CDC37-106 (MRY75), CDC37+ His6-V5-Ste11ΔN (MRY79), and CDC37-106 His6-V5-Ste11ΔN (MRY81) strains.
FIG. 6.
FIG. 6.
Functional interaction between PKA and the Hsp90-chaperone complex. (A) Synthetic lethal interaction between Hsp90 subunit gene STI1 and TPK2. The indicated strains were streaked onto minimal medium agar lacking uracil (−URA) or minimal medium agar containing uracil and 5-fluoroorotic acid (FOA) to select against plasmids expressing URA3 and incubated at 23°C. tpk2-62 sti1Δ (pCDC37-101) (MRY47) and tpk2-62 STI1 (pCDC37-101) [SGY562(pGS224)] strains were used. (B) Sti1 is essential for tpk2(Ts) suppression by CDC37-101. Strains were streaked onto minimal medium agar lacking uracil and incubated at 23°C and 34°C for 2 days. TPK2 sti1Δ CDC37+ (ACY77-3), tpk2-62 sti1Δ CDC37-101 [SGY562(pGS224)], tpk2-62 STI1 CDC37+ [SGY562(pRS316)], and tpk2-62 sti1Δ CDC37-101 (MRY47) strains were used.
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