Activity of Cdc2 and its interaction with the cyclin Cdc13 depend on the molecular chaperone Cdc37 inSchizosaccharomyces pombe

Abstract

Cdc37 is a molecular chaperone whose clients are predominantly protein kinases, many of which are important in cell-cycle progression. Temperature-sensitive mutants of cdc37 in Schizosaccharomyces pombe are lethal at the restrictive temperature, arresting cell division within a single cell cycle. These mutant cells elongate during incubation at the restrictive temperature, consistent with a cell-cycle defect. The cell-cycle arrest arises from defective function of the mutant Cdc37 proteins rather than a reduction in Cdc37 protein levels. Around 80% of the arrested, elongated cells contain a single nucleus and replicated (2C) DNA content, indicating that these mutants arrest the cell cycle in G2 or mitosis (M). Cytological observations show that the majority of cells arrest in G2. In fission yeast, a G2 cell-cycle arrest can arise by inactivation of the cyclin-dependent kinase (Cdk) Cdc2 that regulates entry into mitosis. Studies of the cdc37 temperature-sensitive mutants show a genetic interaction with some cdc2 alleles and overexpression of cdc2 rescues the lethality of some cdc37 alleles at the restrictive temperature, suggesting that Cdc2 is a likely client for the Cdc37 molecular chaperone. In cdc37 temperature-sensitive mutants at the restrictive temperature, the level of Cdc2 protein remains constant but Cdc2 protein kinase activity is greatly reduced. Inactivation of Cdc2 appears to result from the inability to form complexes with its mitotic cyclin partner Cdc13. Further evidence for Cdc2 being a client of Cdc37 in S. pombe comes from the identification of genetic and biochemical interactions between these proteins.

The Schizosaccharomyces pombe Cdc7 Protein Kinase Required for Septum Formation Is a Client Protein of Cdc37

“…In contrast, the Cdc7 kinase activity of a cdc37 ϩ strain remained constant for 3 h at restrictive temperature. One consequence of shifting cdc37ts strains to restrictive temperature is that they arrest in G 2 because of compromised Cdc2 function (37). It was conceivable that the observed fall in Cdc7 kinase activity might be a consequence of reduced Cdc2 kinase activity and/or G 2 arrest, rather than a direct effect of the loss of Cdc37 function.…”

“…Protein extraction, Western blotting, immunoprecipitation, and Cdc7 kinase assays were carried out essentially as described previously (37). The buffer for preparing native protein extracts and carrying out immunoprecipitation experiments was 50 mM Tris-HCl (pH 7.2), 1ϫ Complete Inhibitor (Roche), 1 mM phenylmethylsulfonyl fluoride, 0.1 mM sodium orthovanadate, 0.1 mM EDTA, 1 mM dithiothreitol, 60 mM ␤-glycerophosphate, and 1% (vol/vol) NP-40.…”

“…At restrictive temperature, S. pombe temperaturesensitive cdc37 (cdc37ts) mutants show greatly reduced Cdc2 kinase activity and consequent cell cycle arrest, predominantly in G 2 . Phosphorylation of Cdc2 Tyr15 is not detectably affected in cdc37ts mutants, but the association of Cdc2 with Cdc13 is greatly reduced, accounting for the reduction in kinase activity (37).…”

“…Interestingly, the region of Cdc37 believed to interact with Hsp90 does not appear to be essential for function and cell viability in either yeast (15,36). Two likely clients of Cdc37-Cdc2 (see below) and a signaling kinase, Spc1 (also called Sty1) of the mitogen-activated protein kinase family-have been shown to interact directly with Cdc37 (34,37).…”

“…Several Cdc37 candidate clients were identified in S. cerevisiae as showing synthetic lethality with a cdc37ts mutant (19). We previously observed that S. pombe cdc37 temperature-sensitive mutations interact lethally with temperature-sensitive mutations in swo1 (Hsp90) (36), cdc2 (37), and orb5 (I. Martin, unpublished observation), which encodes casein kinase II (31). In the present study, our aim was to identify new client proteins and/or cochaperones of Cdc37 by carrying out a genetic screen for mutations showing synthetic lethality with a cdc37ts mutation at a temperature permissive for this mutation alone.…”