Inhibiting Akt

Abstract

The Akt family of protein kinases is involved in mediating cell survival signals, and cancers are often associated with mutations in genes encoding negative regulators of Akt activity, such as the lipid phosphatase PTEN. Activation of Akt involves recruitment to the plasma membrane, followed by phosphorylation by activating kinases. Cinar et al . report that the kinases Mst1 and Mst2 inhibit Akt activity in a mechanism that is independent of the kinase activity of Mst. Fractionation of lipid rafts (Triton X-100-insoluble membranes) showed that ~10% of Akt1 was present in this compartment in the prostate cancer cell line LNCaP (a PTEN-deficient cell line in which Akt1 is constitutively active), and coimmunoprecipitation followed by mass spectrometry analysis revealed that Mst1 (also known as STK4) was in a complex with Akt1 in this compartment. The interaction appeared to be direct in that purified proteins interacted. Mst1 is cleaved by caspase into a C-terminal domain containing the regulatory region (Mst1-C) and a N-terminal domain containing the kinase domain (Mst1-N). Full-length Mst1, Mst1-C, and Mst1-N interacted with a tagged form of Akt1 when overexpressed in HEK 293T cells. A kinase-inactive form of the Mst1-N also interacted with Akt1, suggesting that kinase activity was not required for the interaction. All three forms of Mst1 interacted with the C-terminal hydrophobic domain of Akt1 based on experiments with Akt1 mutants. Proteins expressed in transfected cells [Mst1, Mst1-N, and Mst1-C and GFP-Myr-Akt1 (myristoylated and tagged with green fluorescent protein)] and in LNCaP cells (endogenous phosphorylated Akt1 and transfected Mst1-N and Mst1-C) showed that although the full-length Mst1 and Mst1-C colocalized with Akt1 at the plasma membrane and cytoplasm, Mst1-N did not colocalize with Akt1 at the membrane; instead, in the LNCaP cells it colocalized with Akt1 in the nucleus. RNA interference experiments with LNCaP cells showed that depletion of either Mst1 or the related protein Mst2 enhanced Akt1 activity, whereas overexpression of the Mst proteins inhibited Akt1 activity. Expression of Mst1, Mst1-C, Mst1-N, or the kinase-inactive Mst1-N reverted a gastrulation defect induced by expression of constitutively active Akt (Myr-Akt1) in zebrafish embryos, further confirming the negatively regulatory role of Mst. Finally, immunohistochemical analysis of a human prostate cancer tissue array showed that tissues with the highest levels of activated Akt had the lowest levels of Mst1, suggesting that loss of this inhibitory input may be clinically relevant during cancer progression. B. Cinar, P.-K. Fang, M. Lutchman, D. Di Vizio, R. M. Adam, N. Pavlova, M. A. Rubin, P. C. Yelick, M. R. Freeman, The pro-apoptotic kinase Mst1 and its caspase cleavage products are direct inhibitors of Akt1. EMBO J . 26 , 4523-4534 (2007). [PubMed]