Involvement of Hsp90 in Signaling and Stability of 3-Phosphoinositide-dependent Kinase-1

Abstract

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.The characterization of the survival signal transduction pathways stimulated by growth factors and cytokines has revealed that phosphatidylinositide-3-OH kinase (PI3K) 1 is involved in the pathway (1-3). PI3K is a heterodimeric lipid kinase, which consists of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. After stimulation with growth factors and cytokines, PI3K is activated by the interaction of the 85-kDa subunit with phosphotyrosines of activated intracellular domain of growth factor receptors or with the receptorassociated adapter proteins. Subsequently, PI3K associates with the plasma membrane where the 110-kDa catalytic subunit phosphorylates phosphoinositides. The generated phospholipid second messenger molecule, phosphatidylinositol 3,4,5-triphosphate (PtdIns(3,4,5)P 3 ), raises a diverse set of cellular responses (4 -6). The major targets of PtdIns(3,4,5)P 3 are pleckstrin homology (PH) domain-containing proteins, including serine/threonine kinase Akt (also known as protein kinase B (PKB) or RAC-PK) (7, 8).The interaction of PtdIns(3,4,5)P 3 with the PH domain of Akt recruits Akt to the plasma membrane, where it is phosphorylated at two key regulatory residue sites, Thr 308 and Ser 473 . Phosphorylation at both residues is necessary for full activation of Akt and the subsequent regulation of many PI3K-regulated biological responses, including glucose uptake, protein synthesis, and apoptosis inhibition (7,8). Akt phosphorylation at Thr 308 is catalyzed by the ubiquitously expressed 3-phosphoinositide-dependent protein kinase-1 (PDK1) (9 -11). The kinase responsible for phosph...

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