Client protein activation by Hsp90 involves a plethora of cochaperones whose roles are poorly defined. A ubiquitous family of stress-regulated proteins have been identified (Aha1, activator of Hsp90 ATPase) that bind directly to Hsp90 and are required for the in vivo Hsp90-dependent activation of clients such as v-Src, implicating them as cochaperones of the Hsp90 system. In vitro, Aha1 and its shorter homolog, Hch1, stimulate the inherent ATPase activity of yeast and human Hsp90. The identification of these Hsp90 cochaperone activators adds to the complex roles of cochaperones in regulating the ATPase-coupled conformational changes of the Hsp90 chaperone cycle.

No abstract

ERK5 is a mitogen-activated protein (MAP) kinase regulated in human cells by diverse mitogens and stresses but also suspected of mediating the effects of a number of oncogenes. Its expression in the slt2⌬ Saccharomyces cerevisiae mutant rescued several of the phenotypes caused by the lack of Slt2p (Mpk1p) cell integrity MAP kinase. ERK5 is able to provide this cell integrity MAP kinase function in yeast, as it is activated by the cell integrity signaling cascade that normally activates Slt2p and, in its active form, able to stimulate at least one key Slt2p target (Rlm1p, the major transcriptional regulator of cell wall genes). In vitro ERK5 kinase activity was abolished by Hsp90 inhibition. ERK5 activity in vivo was also lost in a strain that expresses a mutant Hsp90 chaperone. Therefore, human ERK5 expressed in yeast is an Hsp90 client, despite the widely held belief that the protein kinases of the MAP kinase class are non-Hsp90-dependent activities. Two-hybrid and protein binding studies revealed that strong association of Hsp90 with ERK5 requires the dual phosphorylation of the TEY motif in the MAP kinase activation loop. These phosphorylations, at positions adjacent to the Hsp90-binding surface recently identified for a number of protein kinases, may cause a localized rearrangement of this MAP kinase region that leads to creation of the Hsp90-binding surface. Complementation of the slt2⌬ yeast defect by ERK5 expression establishes a new tool with which to screen for novel agonists and antagonists of ERK5 signaling as well as for isolating mutant forms of ERK5.Mitogen-activated protein kinase (MAPK) modules consist of 3 protein kinases that stimulate each other in series (MAP3K 3 MAP2K 3 MAPK), resulting in the activation of the terminal and often multifunctional MAPK. These signaling cascades are key components of the highly interactive protein kinase networks in eukaryotic cells. As their name implies, some MAPKs initiate a proliferative response. Others control pathways of embryogenesis, differentiation, stress responses, and cell death (42). Association of MAPK modules with scaffold proteins appears to be one way of ensuring that each MAPK only becomes activated in response to the correct extracellular stimuli or stress signals (52). Once activated, this MAPK can then proceed to phosphorylate its substrates, the latter being often involved in both short-term and longer-term (e.g., transcription-mediated) cellular changes. Many MAPKs have substrates in both the cytoplasm and the nucleus, such that nuclear import/export mechanisms frequently govern their accessibility to their substrates. The final outcome of any MAPK activation event is presumably dictated by this substrate availability in any given cell type, by an intrinsic substrate specificity directed by the docking interaction of the MAPK with its substrates, and by signal attenuation. The latter involves the intervention of the protein phosphatases that, by dephosphorylating and thereby deactivating the MAPK, modulate both the intensity and the dura...

Heat shock protein 90 (Hsp90) is a molecular chaperone required for the activity of many of the most important regulatory proteins of eukaryotic cells (the Hsp90 ‘clients’). Vertebrates have two isoforms of cytosolic Hsp90, Hsp90α and Hsp90β. Hsp90β is expressed constitutively to a high level in most tissues and is generally more abundant than Hsp90α, whereas Hsp90α is stress‐inducible and overexpressed in many cancerous cells. Expressed as the sole Hsp90 of yeast, human Hsp90α and Hsp90β are both able to provide essential Hsp90 functions. Activations of certain Hsp90 clients (heat shock transcription factor, v‐src) were more efficient with Hsp90α, rather than Hsp90β, present in the yeast. In contrast, activation of certain other clients (glucocorticoid receptor; extracellular signal‐regulated kinase‐5 mitogen‐activated protein kinase) was less affected by the human Hsp90 isoform present in these cells. Remarkably, whereas expression of Hsp90β as the sole Hsp90 of yeast rendered cells highly sensitive to the Hsp90 inhibitor radicicol, comparable expression of Hsp90α did not. This raises the distinct possibility that, also for mammalian systems, alterations to the Hsp90α/Hsp90β ratio (as with heat shock) might be a significant factor affecting cellular susceptibility to Hsp90 inhibitors.

Yeast is rendered temperature sensitive with loss of the C-terminal (CT) domain of heat shock transcription factor (Hsf1). This domain loss was found to abrogate heat stimulation of Slt2 (Mpk1), the mitogen-activated protein kinase that directs the reinforced cell integrity gene expression needed for high-temperature growth. In Hsf1 CT domain-deficient cells, Slt2 still undergoes Mkk1/2-directed dual-Thr/Tyr phosphorylation in response to the heat stimulation of cell integrity pathway signaling, but the low Hsp90 expression level suppresses any corresponding increase in Slt2 kinase activity due to Slt2 being a "client" of the Hsp90 chaperone. A non-Hsf1-directed Hsp90 overexpression restored the heat induction of Slt2 activity in these cells, as well as both Slt2-dependent (Rlm1, Swi4) and Slt2-independent (MBF) transcriptional activities. Their high-temperature growth was also rescued, not just by this Hsp90 overexpression but by osmotic stabilization, by the expression of a Slt2-independent form of the Rlm1 transcriptional regulator of cell integrity genes, and by a multicopy SLT2 gene vector. In providing the elevated Hsp90 needed for an efficient activation of Slt2, heat activation of Hsf1 indirectly facilitates (Slt2-directed) heat activation of yet another transcription factor (Rlm1). This provides an explanation as to why, in earlier transcript analysis compared to chromatin immunoprecipitation studies, many more genes of yeast displayed an Hsf1-dependent transcriptional activation by heat than bound Hsf1 directly. The levels of Hsp90 expression affecting transcription factor regulation by Hsp90 client protein kinases also provides a mechanistic model for how heat shock factor can influence the expression of several non-hsp genes in higher organisms.The heat shock response is a stress response almost universally present among living organisms (reviewed in references 36 and 49). In eukaryotic cells, the transcriptional events of this response are due mainly to heat shock transcription factor (HSF). In vitro studies using the purified, recombinant HSFs of Drosophila melanogaster and Saccharomyces cerevisiae have indicated that HSF can directly sense changes to the temperature and the oxidative state within cells (28,59). Mammalian HSF1 undergoes a reversible formation of two redox-sensitive disulfide bonds in response to heat and hydrogen peroxide, an intramolecular bonding that is associated with the homotrimerization of this transcription factor (2). Formation of these HSF1 homotrimers leads, in turn, to this HSF1 undergoing nuclear import and acquiring its DNA binding activity (37). The levels of molecular chaperones are yet another important control over the activity of HSF1 (49).Higher organisms generally have more than one form of HSF (37). In contrast, just a single, essential HSF (Hsf1) is present in yeasts (9). The S. cerevisiae Hsf1 is regulated rather differently than the heat shock-responsive HSF1 of mammals, being constitutively homotrimerized and devoid of the redoxsensitive sulfhydryl groups ...