The mechanism of client protein activation by Hsp90 is enigmatic, and it is uncertain whether Hsp90 employs a common route for all proteins. Using a mutational analysis approach, we investigated the activation of two types of client proteins, glucocorticoid receptor (GR) and the kinase v-Src by the middle domain of Hsp90 (Hsp90M) in vivo. Remarkably, the overall cellular activity of v-Src was highly elevated in a W300A mutant yeast strain due to a 10-fold increase in cellular protein levels of the kinase. In contrast, the cellular activity of GR remained almost unaffected by the W300A mutation but was dramatically sensitive to S485Y and T525I exchanges. In addition, we show that mutations S485Y and T525I in Hsp90M reduce the ATP hydrolysis rate, suggesting that Hsp90 ATPase is more tightly regulated than assumed previously. Therefore, the activation of GR and v-Src has various demands on Hsp90 biochemistry and is dependent on separate functional regions of Hsp90M. Thus, Hsp90M seems to discriminate between different substrate types and to adjust the molecular chaperone for proper substrate activation.Heat shock protein 90 (Hsp90) is a highly conserved, abundant and constitutively expressed homodimeric molecular chaperone of the eukaryotic cytosol. It is specifically involved in the folding and conformational regulation of a limited subset of client proteins. Many natural substrates of Hsp90 are medically relevant signal transduction molecules, e.g., the nuclear receptors for steroid hormones and several kinases, some of them with oncogenic potential (19,23,24). To fulfill its biological function, Hsp90 cooperates with different cochaperones, such as Hop, p50, p23, Aha1, the immunophilins, and others, and acts as part of a multichaperone machine together with Hsp70.Hsp90 is composed of a N-terminal nucleotide binding domain (Hsp90N), a middle domain (Hsp90M), and a C-terminal domain (Hsp90C) that mediates the dimerization of the protein. A hallmark of the Hsp90 reaction cycle is binding and hydrolysis of ATP (20,21,26,34). Although the catalytic center for this reaction has been identified within the N-terminal domain of the protein, the interplay between this part and the other domains of Hsp90 during substrate activation is poorly understood. Emerging evidence suggests that the middle domain of Hsp90 plays an important role in this process. For example, it has been shown that Hsp90M interacts with Aha1, a cochaperone that stimulates Hsp90's rate of ATP hydrolysis and increases the efficiency of client protein activity (8,11,22). Moreover, communication between the middle and N-terminal domains of Hsp90 is essential in vivo (13), probably due to the role of a Hsp90M segment in the proper orientation of the ␥-phosphate group of ATP for hydrolysis by the N-terminal catalytic domain (15). Furthermore, a peptide spanning 14 amino acid residues within Hsp90M has been suggested as the binding site for a natural client protein (30). Several point mutations within Hsp90M that exhibit temperature-sensitive growth defects...
The yeast Saccharomyces cerevisiae utilizes rapidly responding mitogen-activated protein kinase (MAPK) signaling cascades to adapt efficiently to a changing environment. Here we report that phosphorylation of Cdc37p, an Hsp90 cochaperone, by casein kinase 2 controls the functionality of two MAPK cascades in yeast. These pathways, the high-osmolarity glycerol (HOG) pathway and the cell integrity (protein kinase C) MAPK pathway, mediate adaptive responses to high osmotic and cell wall stresses, respectively. Mutation of the phosphorylation site Ser14 in Cdc37p renders cells sensitive to osmotic stress and cell wall perturbation by calcofluor white. We found that levels of the MAPKs Hog1p and Slt2p (Mpk1p) in cells are reduced in a cdc37-S14A mutant, and consequently downstream responses mediated by Hog1p and Slt2p are compromised. Furthermore, we present evidence that Hog1p and Slt2p both interact in a complex with Cdc37p in vivo, something that has not been reported previously. The interaction of Hsp90, Slt2p, and Hog1p with Cdc37p depends on the phosphorylation status of Cdc37p. In fact, our biochemical data show that the osmosensitive phenotype of the cdc37-S14A mutant is due to the loss of the interaction between Cdc37p, Hog1p, and Hsp90. Likewise, during cell wall stress, the interaction of Slt2p with Cdc37p and Hsp90 is crucial for Slt2p-dependent downstream responses, such as the activation of the transcription factor Rlm1p. Interestingly, phosphorylated Slt2p, but not phosphorylated Hog1p, has an increased affinity for Cdc37p. Together these observations suggest that Cdc37p acts as a regulator of MAPK signaling.
Cdc37p, the p50 homolog of Saccharomyces cerevisiae, is an Hsp90 cochaperone involved in the targeting of protein kinases to Hsp90. Here we report a role for Cdc37p in osmoadaptive signalling in this yeast. The osmosensitive phenotype that is displayed by the cdc37-34 mutant strain appears not to be the consequence of deficient signalling through the high osmolarity glycerol (HOG) MAP kinase pathway. Rather, Cdc37p appears to play a role in the filamentous growth (FG) pathway, which mediates adaptation to high osmolarity parallel to the HOG pathway. The osmosensitive phenotype of the cdc37-34 mutant strain is aggravated upon the deletion of the HOG gene. We report that the hyper-osmosensitive phenotype of the cdc37-34, hog1 mutant correlates to a reduced of activity of the FG pathway. We utilized this phenotype to isolate suppressor genes such as KSS1 that encodes a MAP kinase that functions in the FG pathway. We report that Kss1p interacts physically with Cdc37p. Like Kss1p, the second suppressor that we isolated, Dse1p, is involved in cell wall biogenesis or maintenance, suggesting that Cdc37p controls osmoadapation by regulating mitogen-activated protein kinase signalling aimed at adaptive changes in cell wall organization.
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