Hsp90 Phosphorylation Research Articles

Domain mapping studies reveal that the M domain of hsp90 serves as a molecular scaffold to regulate Akt-dependent phosphorylation of endothelial nitric oxide synthase and NO release.

Protein-protein interactions with the molecular chaperone hsp90 and phosphorylation on serine 1179 by the protein kinase Akt leads to activation of endothelial nitric oxide synthase. However, the interplay between these protein-protein interactions remains to be established. In the present study, we show that vascular endothelial growth factor stimulates the coordinated association of hsp90, Akt, and resultant phosphorylation of eNOS. Characterization of the domains of hsp90 required to bind eNOS, using yeast 2-hybrid, cell-based coprecipitation experiments, and GST-fusion proteins, revealed that the M region of hsp90 interacts with the amino terminus of eNOS and Akt. The addition of purified hsp90 to in vitro kinase assays facilitates Akt-driven phosphorylation of recombinant eNOS protein, but not a short peptide encoding the Akt phosphorylation site, suggesting that hsp90 may function as a scaffold for eNOS and Akt. In vivo, coexpression of adenoviral or the cDNA for hsp90 with eNOS promotes nitric oxide release; an effect eliminated using a catalytically functional phosphorylation mutant of eNOS. These results demonstrate that stimulation of endothelial cells with vascular endothelial growth factor recruits eNOS and Akt to an adjacent region on the same domain of hsp90, thereby facilitating eNOS phosphorylation and enzyme activation.

Purification and characterization of porcine testis 90-kDa heat shock protein (HSP90) as a substrate for various protein kinases.

We purified a large quantity of HSP90 from porcine testis by hydroxylapatite (HA-HSP90) and SDS-PAGE/electroelution (eluted-HSP90) to explore the molecular mechanism of HSP90 phosphorylation affecting its metabolism. The purified HSP90 was used as an antigen to raise polyclonal antibodies in rabbits. Immunoblot analysis revealed that most purified HSP90 was HSP90alpha. Compared with the commercial anti-HSP90 antibody, the polyclonal antibody raised in this study could specifically detect the testis HSP90 and immunoprecipitate HSP90 from tissue homogenates or cell extracts. Incubation of the purified HSP90 or HSP90 immunoprecipitated from extracts of human A431 cells, Balb/c 3T3 fibroblasts, and porcine testis with [gamma-32P]ATP/Mg2+ resulted in phosphorylation of HSP90. However, the eluted-HSP90 lost its phosphorylation ability when incubated with [gamma-32P]ATP x Mg2+ alone but could be phosphorylated by various protein kinases, including PKA, CKII, kinase FA/GSK-3 alpha, and AK. The order of phosphorylation of HSP90 by these kinases is PKA = CKII > AK >> kinase FA/GSK-3 alpha.

Hsp90 Phosphorylation Is Linked to Its Chaperoning Function: ASSEMBLY OF THE REOVIRUS CELL ATTACHMENT PROTEIN

Studies on Hsp90 have mainly focused on its involvement in the activation of several families of protein kinases and of steroid hormone receptors. Little is known regarding the role of Hsp90 in the folding of nascent proteins. We previously reported that Hsp90 plays an active role in the posttranslational assembly of the C-terminal globular head of the reovirus attachment protein final sigma1. We show here that Hsp90 becomes phosphorylated in this process. However, only the unphosphorylated form of Hsp90 is complexed with final sigma1, suggesting that Hsp90 phosphorylation is coupled to the release of the chaperone from the target protein. Geldanamycin, which blocks final sigma1 maturation by preventing the release of Hsp90 from final sigma1, also inhibits Hsp90 phosphorylation. Taken together, these results demonstrate that Hsp90 phosphorylation is linked to its chaperoning function.

Casein kinase II is implicated in the regulation of heme-controlled translational inhibitor of reticulocyte lysates.

The hemin-controlled inhibitor, or HCI, is a cyclic nucleotide-independent protein kinase which specifically phosphorylates the alpha subunit of eukaryotic initiation factor 2 (eIF-2) leading to potential limitations in functional eIF-2 and decreases in protein synthesis initiation. We have recently demonstrated that hemin regulates eIF-2 alpha kinase activity by promoting formation of the inactive heterodimer between HCI and the 90-kDa heat shock protein (hsp90). The formation of the inactive form of HCI by hemin is prevented by treatment with sulfhydryl reagents such as N-ethylmaleimide or when hsp90 is previously phosphorylated (Méndez, R., Moreno, A., and de Haro, C. (1992) J. Biol. Chem. 267, 11500-11507). In this report, using isoelectric focusing and Western blot analyses with antibodies against a synthetic HCI peptide, we have demonstrated that HCI was also phosphorylated when a heme-reversible HCI preparation was preincubated with ATP. Furthermore, our results indicate that casein kinase II (CK II) is the enzyme involved in the regulation of HCI. Thus, the synthetic peptide RRREEETEEE, which is a specific substrate for CK II, acts as a competitive inhibitor of HCI and hsp90 phosphorylation and, at the same time, inhibits the activation of HCI, whereas a synthetic peptide which corresponds to residues 45-59 of the alpha subunit of eIF-2, including the Ser51 phosphorylated by HCI, only inhibits competitively the phosphorylation of eIF-2 alpha. In addition, treatments which modify hsp90 disabling the formation of the inactive dimer with HCI make unnecessary the presence of CK II for activation of HCI. The data strongly suggest that hemin promotes formation of an inactive HCI.hsp90 dimer preventing phosphorylation by CK II. Interestingly, the addition of the CK II peptide substrate also prevents the activation of HCI in a heme-deficient reticulocyte lysate. We hypothesize, therefore, that under physiological conditions, CK II activity appears to be necessary for activation of HCI.

Estradiol increases phosphorylation of the 90 kDa heat shock protein not associated with estradiol receptor in MCF-7 cells in culture

MCF-7 cells in monolayer culture were incubated with [ 32P]orthophosphate for 18 h followed by covalent whole cell labelling of the estradiol receptor with tritiated tamoxifen aziridine ([ 3H]TA). The heat shock protein (hsp-90) bound to receptor was precipitated with monoclonal antibodies H222 or JS 34/32, coupled to protein A-Sepharose and purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions. Hsp-90 not associated with receptor was similarly purified after isolation with the monoclonal antibody AC88. It was found that estradiol treatment of the cells markedly increased phosphate incorporation in the free hsp-90, without affecting heat shock protein bound to receptor. A 6-fold increase in phosphate content was observed after 10 min incubation of the cells with estradiol. A similar effect was seen after treatment of the cells with the phorbol ester 12- O-tetrade-canoylphorbol 13-acetate (TPA). The calcium ionophore A23187 had no influence on hsp-90 phosphorylation, and treatment of the cells with forskolin to increase the cellular content of cAMP had a reverse effect. A 50% reduction of the phosphate content in the free hsp-90 was observed after 15 min treatment. The observation that estradiol, TPA and forskolin had effect only on hsp-90 not bound to receptor is an indication that the receptor-hsp-90 complex exists in vivo. Time course studies show that the effect of estradiol is non-genomic. Two possible explanations of the results seem to exist. Either estradiol induces an increase in the degree of phosphorylation of hsp-90, or hsp-90 is translocated to the cytosol from a different cellular compartment.