Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein.

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1.

Inhibition of the phosphatidylinositol 3-kinase/Akt pathway by inositol pentakisphosphate results in antiangiogenic and antitumor effects.

The purpose of this study was to investigate the antiangiogenic and in vivo properties of the recently identified phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor Inositol(1,3,4,5,6) pentakisphosphate [Ins(1,3,4,5,6)P5]. Because activation of the PI3K/Akt pathway is a crucial step in some of the events leading to angiogenesis, the effect of Ins(1,3,4,5,6)P5 on basic fibroblast growth factor (FGF-2)-induced Akt phosphorylation, cell survival, motility, and tubulogenesis in vitro was tested in human umbilical vein endothelial cells (HUVEC). The effect of Ins(1,3,4,5,6)P5 on FGF-2-induced angiogenesis in vivo was evaluated using s.c. implanted Matrigel in mice. In addition, the effect of Ins(1,3,4,5,6)P5 on growth of ovarian carcinoma SKOV-3 xenograft was tested. Here, we show that FGF-2 induces Akt phosphorylation in HUVEC resulting in antiapoptotic effect in serum-deprived cells and increase in cellular motility. Ins(1,3,4,5,6)P5 blocks FGF-2-mediated Akt phosphorylation and inhibits both survival and migration in HUVEC. Moreover, Ins(1,3,4,5,6)P5 inhibits the FGF-2-mediated capillary tube formation of HUVEC plated on Matrigel and the FGF-2-induced angiogenic reaction in BALB/c mice. Finally, Ins(1,3,4,5,6)P5 blocks the s.c. growth of SKOV-3 xenografted in nude mice to the same extent than cisplatin and it completely inhibits Akt phosphorylation in vivo. These data definitively identify the Akt inhibitor Ins(1,3,4,5,6)P5 as a specific antiangiogenic and antitumor factor. Inappropriate activation of the PI3K/Akt pathway has been linked to the development of several diseases, including cancer, making this pathway an attractive target for therapeutic strategies. In this respect, Ins(1,3,4,5,6)P5, a water-soluble, natural compound with specific proapoptotic and antiangiogenic properties, might result in successful anticancer therapeutic strategies.