Genetic screens in Drosophila have identified p50(cdc37) to be an essential component of the sevenless receptor/mitogen-activated kinase protein (MAPK) signaling pathway, but neither the function nor the target of p50(cdc37) in this pathway has been defined. In this study, we examined the role of p50(cdc37) and its Hsp90 chaperone partner in Raf/Mek/MAPK signaling biochemically. We found that coexpression of wild-type p50(cdc37) with Raf-1 resulted in robust and dose-dependent activation of Raf-1 in Sf9 cells. In addition, p50(cdc37) greatly potentiated v-Src-mediated Raf-1 activation. Moreover, we found that p50(cdc37) is the primary determinant of Hsp90 recruitment to Raf-1. Overexpression of a p50(cdc37) mutant which is unable to recruit Hsp90 into the Raf-1 complex inhibited Raf-1 and MAPK activation by growth factors. Similarly, pretreatment with geldanamycin (GA), an Hsp90-specific inhibitor, prevented both the association of Raf-1 with the p50(cdc37)-Hsp90 heterodimer and Raf-1 kinase activation by serum. Activation of Raf-1 via baculovirus coexpression with oncogenic Src or Ras in Sf9 cells was also strongly inhibited by dominant negative p50(cdc37) or by GA. Thus, formation of a ternary Raf-1-p50(cdc37)-Hsp90 complex is crucial for Raf-1 activity and MAPK pathway signaling. These results provide the first biochemical evidence for the requirement of the p50(cdc37)-Hsp90 complex in protein kinase regulation and for Raf-1 function in particular.

Interferons (IFNs) inhibit cell growth in a Stat1-dependent fashion that involves regulation of c-myc expression. IFN-gamma suppresses c-myc in wild-type mouse embryo fibroblasts, but not in Stat1-null cells, where IFNs induce c-myc mRNA rapidly and transiently, thus revealing a novel signaling pathway. Both tyrosine and serine phosphorylation of Stat1 are required for suppression. Induced expression of c-myc is likely to contribute to the proliferation of Stat1-null cells in response to IFNs. IFNs also suppress platelet-derived growth factor (PDGF)-induced c-myc expression in wild-type but not in Stat1-null cells. A gamma-activated sequence element in the promoter is necessary but not sufficient to suppress c-myc expression in wild-type cells. In PKR-null cells, the phosphorylation of Stat1 on Ser727 and transactivation are both defective, and c-myc mRNA is induced, not suppressed, in response to IFN-gamma. A role for Raf-1 in the Stat1-independent pathway is revealed by studies with geldanamycin, an HSP90-specific inhibitor, and by expression of a mutant of p50(cdc37) that is unable to recruit HSP90 to the Raf-1 complex. Both agents abrogated the IFN-gamma-dependent induction of c-myc expression in Stat1-null cells.

Recent studies indicate that p50cdc37 facilitates Hsp90-mediated biogenesis of certain protein kinases. In this report, we examined whether p50 cdc37 is required for the biogenesis of the heme-regulated eIF2␣ kinase (HRI) in reticulocyte lysate. p50 cdc37 interacted with nascent HRI co-translationally and this interaction persisted during the maturation and activation of HRI. p50 cdc37 stimulated HRI's activation in response to heme deficiency, but did not activate HRI per se. p50 cdc37 function was specific to immature and inactive forms of the kinase. Analysis of mutant Cdc37 gene products indicated that the N-terminal portion of p50 cdc37 interacted with immature HRI, but not with Hsp90, while the C-terminal portion of p50 cdc37 interacted with Hsp90. The Hsp90-specific inhibitor geldanamycin disrupted the ability of both Hsp90 and p50 cdc37 to bind HRI and promote its activation, but did not disrupt the native association of p50 cdc37 with Hsp90. A C-terminal truncated mutant of p50 cdc37 inhibited HRI's activation, prevented the interaction of Hsp90 with HRI, and bound to HRI irrespective of geldanamycin treatment. Additionally, native complexes of HRI with p50 cdc37 were detected in cultured K562 erythroleukemia cells. These results suggest that p50 cdc37 provides an activity essential to HRI biogenesis via a process regulated by nucleotide-mediated conformational switching of its partner Hsp90.The heme-regulated inhibitor (HRI) 1 of protein synthesis is a protein-serine kinase which coordinates the synthesis of globin chains with the availability of heme in reticulocytes (reviewed in Refs. 1 and 2). Under heme-deficient conditions, HRI phosphorylates the ␣-subunit of eukaryotic translational initiation factor eIF2. Phosphorylation of eIF2␣ causes an inhibition of polypeptide chain initiation and the arrest of protein synthesis, preventing the synthesis of apo-globin chains in the absence of heme. HRI is also activated under heme-replete conditions in response to a host of other adverse environmental stimuli, such as heat shock, agents that generate oxidative stress, and the presence of denatured proteins (1, 2).The biogenesis and activation of HRI into an active hemeregulatable eIF2␣ kinase requires its functional interaction with the chaperone machinery containing the 90-kDa heat shock protein (Hsp90) and the 70-kDa heat shock cognate protein (Hsc70) (3, 4). During HRI biogenesis and its subsequent transformation and activation, several discrete HRI intermediates are generated; these intermediates can be distinguished on the basis of their competence to become an active kinase in response to heme deficiency or upon treatment with sulfhydryl reactive compounds such as N-ethylmaleimide. Immediately after their synthesis, HRI molecules are not active in hemereplete or heme-deficient rabbit reticulocyte lysate (RRL) and cannot be activated by N-ethylmaleimide treatment. This immature population interacts with Hsp90 and Hsc70 (3-7). Subsequent to this immature phase, a "mature-competent" HRI population appear...

Heat shock factor 1 (HSF1) regulates the transcription of molecular chaperone hsp genes. However, the cellular control mechanisms that regulate HSF1 activity are not well understood. In this study, we have demonstrated for the first time that human HSF1 binds to the essential cell signaling protein 14-3-3. Binding of HSF1 to 14-3-3 occurs in cells in which extracellular signal regulated kinase (ERK) is activated and blockade of the ERK pathway by treatment with the specific ERK pathway inhibitor PD98059 in vivo strongly suppresses the binding. We previously showed that ERK1 phosphorylates HSF1 on serine 307 and leads to secondary phosphorylation by glycogen synthase kinase 3 (GSK3) on serine 303 within the regulatory domain and that these phosphorylation events repress HSF1. We show here that HSF1 binding to 14-3-3 requires HSF1 phosphorylation on serines 303 and 307. Furthermore, the serine phosphorylation-dependent binding of HSF1 to 14-3-3 results in the transcriptional repression of HSF1 and its sequestration in the cytoplasm. Leptomycin B, a specific inhibitor of nuclear export receptor CRM1, was found to reverse the cytoplasmic sequestration of HSF1 mediated by 14-3-3, suggesting that CRM1/14-3-3 directed nuclear export plays a major role in repression of HSF1 by the ERK/GSK3/14-3-3 pathway. Our experiments indicate a novel pathway for HSF1 regulation and suggest a mechanism for suppression of its activity during cellular proliferation.Heat shock transcription factor 1 (HSF1) is the mammalian regulator of the heat shock response and activates the transcription of heat shock protein (Hsp) molecular chaperone genes (43,47,49). Inactivation of the murine hsf1gene has been shown to confer a complex phenotype, indicating an essential function for hsf1 in growth, in development, and in acute response to stress (35). Disruption of hsf1 (i.e., hsf1 Ϫ/Ϫ ) in mouse embryonic fibroblasts leads to a profound loss of thermotolerance and markedly increased susceptibility to heatinduced apoptosis (11,35). hsf1 is required as a maternal factor during the early cleavage stage of development in the Ϫ/Ϫ mouse embryo (11). hsf1-deficient mice can survive to adulthood but display severe defects in the chorioallantoic placenta that result in increased prenatal lethality (56). In addition, the aging process is associated with degeneration of the heat shock response, and transcriptional activity of HSF1 protein was significantly reduced with age in a cell-free system, as well as in isolated hepatocytes (26). Understanding the processes involved in HSF1 regulation may therefore aid in delineating its role in resistance to stress, development, and aging.Under normal conditions, cellular HSF1 exists in a predominantly transcriptionally repressed state (44, 59). Such HSF1 is monomeric, is constitutively phosphorylated, and lacks the ability to bind the cis-acting heat shock elements (HSEs) located in the promoters of Hsp genes (50, 55). Induction of transcriptional activity by heat shock then results in the conversion of HSF1 from in...