Involvement of Hsp90 in signaling and stability of 3-phosphoinositide-dependent kinase-1.

Serine/threonine kinase Akt is thought to mediate many biological actions toward anti-apoptotic responses. Screening of drugs that could interfere with the Akt signaling pathway revealed that Hsp90 inhibitors (e.g. geldanamycin, radicicol, and its analogues) induced Akt dephosphorylation, which resulted in Akt inactivation and apoptosis of the cells. Hsp90 inhibitors did not directly affect Akt kinase activity in vitro. Thus, we examined the effects of Hsp90 inhibitors on upstream Akt kinases, phosphatidylinositide-3-OH kinase (PI3K) and 3-phosphoinositide-dependent protein kinase-1 (PDK1). Hsp90 inhibitors had no effect on PI3K protein expression. In contrast, treatment of the cells with Hsp90 inhibitors decreased the amount of PDK1 without directly inhibiting PDK1 kinase activity. We found that the kinase domain of PDK1 was essential for complex formation with Hsp90 and that Hsp90 inhibitors suppressed PDK1 binding to Hsp90. PDK1 degradation mechanisms revealed that inhibition of PDK1 binding to Hsp90 caused proteasome-dependent degradation of PDK1. Treatment of proteasome inhibitors increased the amount of detergent-insoluble PDK1 in Hsp90 inhibitor-treated cells. Therefore, the association of PDK1 with Hsp90 regulates its stability, solubility, and signaling. Because Akt binding to Hsp90 is also involved in the maintenance of Akt kinase activity, Hsp90 plays an important role in PDK1-Akt survival signaling pathway.     

Roles of 3-phosphoinositide-dependent kinase 1 in the regulation of endothelial nitric-oxide synthase phosphorylation and function by heat shock protein 90

The 90-kDa heat shock protein (Hsp90) plays an important role in endothelial nitric-oxide synthase (eNOS) regulation. Besides acting as an allosteric enhancer, Hsp90 was shown to serve as a module recruiting Akt to phosphorylate the serine 1179/1177 (bovine/human) residue of eNOS. Akt is activated by the phosphorylation of 3-phosphoinositide-dependent kinase 1 (PDK1). Whether PDK1 is involved in the actions of Hsp90 on eNOS phosphorylation and function remains unknown. To address this issue, we treated bovine eNOS stably transfected human embryonic kidney 293 cells with Hsp90 inhibitors and determined the alterations of phospho-eNOS, Akt, and PDK1. Both geldanamycin and radicicol, two structurally different Hsp90 inhibitors, selectively reduced serine 1179-phosphorylated eNOS, leading to decreased enzyme activity. In Hsp90-inhibited cells, eNOS-associated phospho-Akt was decreased, but the total amount of Akt associated with eNOS remained the same. Further studies showed that Hsp90 inhibition dramatically depleted intracellular PDK1. Proteasome but not caspase blockade prevented the loss of PDK1 caused by Hsp90 inhibition. Silencing the PDK1 gene by small interfering RNA was sufficient to induce reduction of phospho-Akt and consequent loss of serine 1179-phosphorylated eNOS. Moreover, overexpression of PDK1, but not Akt, reversed Hsp90 inhibition-induced loss of eNOS serine 1179 phosphorylation and salvaged enzymatic activity. Thus, in addition to functioning as a module to recruit Akt to eNOS, Hsp90 also critically stabilized PDK1 by preventing it from proteasomal degradation. Inhibition of Hsp90 function resulted in PDK1 depletion and thus triggered a cascade of Akt deactivation, loss of eNOS serine 1179 phosphorylation, and decrease of enzyme function.     

Antibiotic radicicol binds to the N-terminal domain of Hsp90 and shares important biologic activities with geldanamycin

The molecular chaperone Hsp90 plays an essential role in the folding and function of important cellular proteins including steroid hormone receptors, protein kinases and proteins controlling the cell cycle and apoptosis. A 15 Å deep pocket region in the N-terminal domain of Hsp90 serves as an ATP/ADP-binding site and has also been shown to bind geldanamycin, the only specific inhibitor of Hsp90 function described to date. We now show that radicicol, a macrocyclic antifungal structurally unrelated to geldanamycin, also specifically binds to Hsp90. Moreover, radicicol competes with geldanamycin for binding to the N-terminal domain of the chaperone, expressed either by in vitro translation or as a purified protein, suggesting that radicicol shares the geldanamycin binding site. Radicicol, as does geldanamycin, also inhibits the binding of the accessory protein p23 to Hsp90, and interferes with assembly of the mature progesterone receptor complex. Radicicol does not deplete cells of Hsp90, but rather increases synthesis as well as the steady-state level of this protein, similar to a stress response. Finally, radicicol depletes SKBR3 cells of p 185^erbB2, Raf-1 and mutant p53, similar to geldanamycin. Radicicol thus represents a structurally unique antibiotic, and the first non-benzoquinone ansamycin, capable of binding to Hsp90 and interfering with its function.     

Radicicol, an inhibitor of Hsp90, enhances TRAIL-induced apoptosis in human epithelial ovarian carcinoma cells by promoting activation of apoptosis-related proteins

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in various cancer cells. Hsp90 is known to be involved in cell survival and growth in tumor cells. Nevertheless, Hsp90 inhibitors exhibit a variable effect on the cytotoxicity of anticancer drugs. Furthermore, the combined effect of Hsp90 inhibitors on TRAIL-induced apoptosis in epithelial ovarian cancer cells has not been determined. To assess the ability of an inhibitor of Hsp90 inhibitor radicicol to promote apoptosis, we investigated the effect of radicicol on TRAIL-induced apoptosis in the human epithelial ovarian carcinoma cell lines OVCAR-3 and SK-OV-3. TRAIL induced a decrease in Bid, Bcl-2, Bcl-xL, and survivin protein levels, increase in Bax levels, loss of the mitochondrial transmembrane potential, cytochrome c release, activation of caspases (-8, -9, and -3), cleavage of PARP-1 and an increase in the tumor suppressor p53 levels. Radicicol enhanced TRAIL-induced apoptosis-related protein activation, nuclear damage and cell death. These results suggest that radicicol may potentiate the apoptotic effect of TRAIL on ovarian carcinoma cell lines by increasing the activation of the caspase-8- and Bid-dependent pathway and the mitochondria-mediated apoptotic pathway, leading to caspase activation. Radicicol may confer a benefit in the TRAIL treatment of epithelial ovarian adenocarcinoma.     

Palmitate modulates intracellular signaling, induces endoplasmic reticulum stress, and causes apoptosis in mouse 3T3-L1 and rat primary preadipocytes

Although fatty acids enhance preadipocyte differentiation in the presence of adequate hormone cocktails, little is known regarding their effects in the absence of these hormones. We have now shown that palmitate, a common long-chain saturated fatty acid, induced apoptosis in both mouse 3T3-L1 and rat primary preadipocytes grown in a normal serum-containing medium. Treatment of preadipocytes with palmitate induced multiple endoplasmic reticulum (ER) stress responses, evidenced by increased protein content of CHOP and GRP78 and splicing of XBP-1 mRNA, as well as altered phosphorylation of eIF2alpha and increased phosphorylation of JNK and Erk1/2. Intriguingly, palmitate induced an early activation of Akt but diminished both Akt activation and its protein mass after prolonged incubation (>6 h). In association with these changes, palmitate reduced expression of beta-catenin and its downstream target, c-Myc and cyclin D1, two key prosurvival proteins. Overexpression of constitutively active Akt did not block the apoptotic effect of palmitate. Cotreatment with unsaturated fatty acids (oleate, linoleate) or with LiCl (a glycogen synthase kinase-3beta inhibitor) attenuated the palmitate-induced apoptosis. Subsequent analysis suggested that the unsaturated fatty acids probably counteracted palmitate by reducing, not eliminating, ER stress, whereas LiCl probably improved viability by activating the Wnt signaling pathway. Cotreatment of palmitate with a standard adipogenic hormone cocktail also abolished the apoptotic effect and promoted adipocyte differentiation. Collectively, our results suggest that palmitate causes multiple cellular stresses that may lead to apoptosis in preadipocytes in the absence of adipogenic stimuli, highlighting the importance of exogenous hormones in directing cell fate in response to increased fatty acid influx.     

Distinct structural mechanisms for inhibition of pyruvate dehydrogenase kinase isoforms by AZD7545, dichloroacetate, and radicicol

Pyruvate dehydrogenase kinase (PDK) isoforms are molecular switches that downregulate the pyruvate dehydrogenase complex (PDC) by reversible phosphorylation in mitochondria. We have determined structures of human PDK1 or PDK3 bound to the inhibitors AZD7545, dichloroacetate (DCA), and radicicol. We show that the trifluoromethylpropanamide end of AZD7545 projects into the lipoyl-binding pocket of PDK1. This interaction results in inhibition of PDK1 and PDK3 activities by aborting kinase binding to the PDC scaffold. Paradoxically, AZD7545 at saturating concentrations robustly increases scaffold-free PDK3 activity, similar to the inner lipoyl domain. Good DCA density is present in the helix bundle in the N-terminal domain of PDK1. Bound DCA promotes local conformational changes that are communicated to both nucleotide-binding and lipoyl-binding pockets of PDK1, leading to the inactivation of kinase activity. Finally, radicicol inhibits kinase activity by binding directly to the ATP-binding pocket of PDK3, similar to Hsp90 and Topo VI from the same ATPase/kinase superfamily.     

Caffeic acid phenethyl ester causes p21 induction, Akt signaling reduction, and growth inhibition in PC-3 human prostate cancer cells

Caffeic acid phenethyl ester (CAPE) treatment suppressed proliferation, colony formation, and cell cycle progression in PC-3 human prostate cancer cells. CAPE decreased protein expression of cyclin D1, cyclin E, SKP2, c-Myc, Akt1, Akt2, Akt3, total Akt, mTOR, Bcl-2, Rb, as well as phosphorylation of Rb, ERK1/2, Akt, mTOR, GSK3α, GSK3β, PDK1; but increased protein expression of KLF6 and p21(Cip1). Microarray analysis indicated that pathways involved in cellular movement, cell death, proliferation, and cell cycle were affected by CAPE. Co-treatment of CAPE with chemotherapeutic drugs vinblastine, paclitaxol, and estramustine indicated synergistic suppression effect. CAPE administration may serve as a potential adjuvant therapy for prostate cancer.     

Mechanisms of activation of eNOS by 20-HETE and VEGF in bovine pulmonary artery endothelial cells

We have demonstrated that VEGF-induced dilation of bovine pulmonary arteries is associated with activation of cytochrome P-450 family 4 (CYP4) enzymes and eNOS. We hypothesized that VEGF and the CYP4 product 20-HETE would trigger common downstream pathways of intracellular signaling to activate eNOS. We treated bovine pulmonary artery endothelial cells (BPAECs) with 20-HETE (1 microM) or VEGF (8.3 nM) and examined three molecular events known to activate eNOS: 1) phosphorylation at serine 1179, 2) phosphorylation of protein kinase B (Akt), which subsequently phosphorylates eNOS, and 3) association of eNOS with 90-kDa heat shock protein (Hsp90). Both 20-HETE and VEGF increase the phosphorylation of eNOS at serine 1179 and Akt at serine 473. The CYP4 inhibitor dibromododecynyl methyl sulfonamide (DDMS) blocks VEGF-induced phosphorylation of eNOS. VEGF had no effect on the binding of Hsp90 with eNOS, whereas 20-HETE decreased the association of the protein partners. Inhibition of Akt-phosphatidylinositol 3-kinase with wortmannin blocks both 20-HETE and VEGF-induced relaxation of pulmonary arteries, supporting the functional contribution of Akt phosphorylation to the vasoactive actions of both agents. Treatment with radicicol had no effect on 20-HETE-induced relaxation of pulmonary arteries, consistent with an absence of effect on association of Hsp90 to eNOS, whereas radicicol partially blocked VEGF-evoked relaxations, possibly secondary to effects on endpoints other than Hsp90 association with eNOS. In conclusion, VEGF and 20-HETE share eNOS activation pathways, including phosphorylation of serine 1179 and phosphorylation of Akt. Unlike aortic endothelial cells, eNOS activation in BPAECs by either VEGF or 20-HETE does not appear to require increased association of Hsp90.     

Interaction of radicicol with members of the heat shock protein 90 family of molecular chaperones.

The Hsp90 family of proteins in mammalian cells consists of Hsp90 alpha and beta, Grp94, and Trap-1 (Hsp75). Radicicol, an antifungal antibiotic that inhibits various signal transduction proteins such as v-src, ras, Raf-1, and mos, was found to bind to Hsp90, thus making it the prototype of a second class of Hsp90 inhibitors, distinct from the chemically unrelated benzoquinone ansamycins. We have used two novel methods to immobilize radicicol, allowing for detailed analyses of drug-protein interactions. Using these two approaches, we have studied binding of the drug to N-terminal Hsp90 point mutants expressed by in vitro translation. The results point to important drug contacts with amino acids inside the N-terminal ATP/ADP-binding pocket region and show subtle differences when compared with geldanamycin binding. Radicicol binds more strongly to Hsp90 than to Grp94, the Hsp90 homolog that resides in the endoplasmic reticulum. In contrast to Hsp90, binding of radicicol to Grp94 requires both the N-terminal ATP/ADP-binding domain as well as the adjacent negatively charged region. Radicicol also specifically binds to yeast Hsp90, Escherichia coli HtpG, and a newly described tumor necrosis factor receptor-interacting protein, Trap-1, with greater homology to bacterial HtpG than to Hsp90. Thus, the radicicol-binding site appears to be specific to and is conserved in all members of the Hsp90 family of molecular chaperones from bacteria to mammals, but is not present in other molecular chaperones with nucleotide-binding domains.     

Caffeic Acid Phenethyl Ester Causes p21Cip1 Induction, Akt Signaling Reduction, and Growth Inhibition in PC-3 Human Prostate Cancer Cells

Caffeic acid phenethyl ester (CAPE) treatment suppressed proliferation, colony formation, and cell cycle progression in PC-3 human prostate cancer cells. CAPE decreased protein expression of cyclin D1, cyclin E, SKP2, c-Myc, Akt1, Akt2, Akt3, total Akt, mTOR, Bcl-2, Rb, as well as phosphorylation of Rb, ERK1/2, Akt, mTOR, GSK3α, GSK3β, PDK1; but increased protein expression of KLF6 and p21^Cip1. Microarray analysis indicated that pathways involved in cellular movement, cell death, proliferation, and cell cycle were affected by CAPE. Co-treatment of CAPE with chemotherapeutic drugs vinblastine, paclitaxol, and estramustine indicated synergistic suppression effect. CAPE administration may serve as a potential adjuvant therapy for prostate cancer.     

Regulation of 3-phosphoinositide-dependent protein kinase-1 (PDK1) by Src involves tyrosine phosphorylation of PDK1 and Src homology 2 domain binding

3-Phosphoinositide-dependent protein kinase-1 (PDK1) appears to play a central regulatory role in many cell signalings between phosphoinositide-3 kinase and various intracellular serine/threonine kinases. In resting cells, PDK1 is known to be constitutively active and is further activated by tyrosine phosphorylation (Tyr(9) and Tyr(373/376)) following the treatment of the cell with insulin or pervanadate. However, little is known about the mechanisms for this additional activation of PDK1. Here, we report that the SH2 domain of Src, Crk, and GAP recognized tyrosine-phosphorylated PDK1 in vitro. Destabilization of PDK1 induced by geldanamycin (a Hsp90 inhibitor) was partially blocked in HEK 293 cells expressing PDK1-Y9F. Co-expression of Hsp90 enhanced PDK1-Src complex formation and led to further increased PDK1 activity toward PKB and SGK. Immunohistochemical analysis with anti-phospho-Tyr(9) antibodies showed that the level of Tyr(9) phosphorylation was markedly increased in tumor samples compared with normal. Taken together, these data suggest that phosphorylation of PDK1 on Tyr(9), distinct from Tyr(373/376), is important for PDK1/Src complex formation, leading to PDK1 activation. Furthermore, Tyr(9) phosphorylation is critical for the stabilization of both PDK1 and the PDK1/Src complex via Hsp90-mediated protection of PDK1 degradation.     

Regulation of kinase activity of 3-phosphoinositide-dependent protein kinase-1 by binding to 14-3-3

3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a central role in activating the protein kinase A, G, and C subfamily. In particular, PDK1 plays an important role in regulating the Akt survival pathway by phosphorylating Akt on Thr-308. PDK1 kinase activity was thought to be constitutively active; however, recent reports suggested that its activity is regulated by binding to other proteins, such as protein kinase C-related kinase-2 (PRK2), p90 ribosomal protein S6 kinase-2 (RSK2), and heat-shock protein 90 (Hsp90). Here we report that PDK1 binds to 14-3-3 proteins in vivo and in vitro through the sequence surrounding Ser-241, a residue that is phosphorylated by itself and is critical for its kinase activity. Mutation of PDK1 to increase its binding to 14-3-3 decreased its kinase activity in vivo. By contrast, mutation of PDK1 to decrease its interaction with 14-3-3 resulted in increased PDK1 kinase activity. Moreover, incubation of wild-type PDK1 with recombinant 14-3-3 in vitro decreased its kinase activity. These data indicate that PDK1 kinase activity is negatively regulated by binding to 14-3-3 through the PDK1 autophosphorylation site Ser-241.