Phosphatidylinositol Ether Lipid Analogues That Inhibit AKT Also Independently Activate the Stress Kinase, p38α, through MKK3/6-independent and -dependent Mechanisms

Joell J Gills,S Sianna Castillo,Chunyu Zhang,Pavel A Petukhov,Regan M Memmott,Melinda Hollingshead,Noel Warfel,Jiahuai Han,Alan P Kozikowski,Phillip A Dennis

doi:10.1074/jbc.m701108200

Abstract

Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38alpha, was uniformly activated 2-fold. Molecular modeling of p38alpha revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38alpha(-/-) cells, we showed that p38alpha is not required for PIA-induced apoptosis but is required for H(2)O(2)- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38alpha(-/-) cells increased apoptosis. These studies indicate that p38alpha is activated by PIAs through a novel mechanism and show that p38alpha activation contributes to PIA-induced cell death. Independent modulation of Akt and p38alpha could account for the profound cytotoxicity of PIAs.

Highlights

We identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity
Because p38 activation at early time points did not seem to Mechanisms of p38 Activation by PIAs—Because the data depend on upstream kinases, we studied whether PIAs could from the purified kinase screen and molecular modeling suggested induce binding of TAB1 to p38, which has other kinases, we assessed the p38 pathway at early time points in previously been shown to induce p38 autophosphorylation in
The usefulness of a targeted agent will depend upon modulation of its desired target as well as other unintended off target effects

Summary

Introduction

We identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. Activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38␣؊/؊ cells increased apoptosis. We used molecular modeling to guide synthesis of phosphatidylinositol ether lipid analogues (PIAs) that were designed to inhibit the pleckstrin homology (PH) domain of Akt. PIAs rapidly inhibited Akt and selectively induced apoptosis in cell lines with high levels of constitutively active Akt [6]. Identification of off target effects could expand possible clinical applications or provide greater understanding of drug-associated toxicities, which is perhaps best illustrated by the experience with imatinib mesylate. The identification of deleterious off target effects could facilitate choosing the best inhibitors of Akt that are most efficacious and least toxic

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Conclusion