Abstract
Understanding the regulation of Akt has been of major interest for elucidating the control of normal cellular physiology as well as malignant transformation. The paradigm for activation of Akt involves phosphatidylinositol 3-kinase-dependent membrane localization followed by activating phosphorylation of Thr-308 and Ser-473. Many of the activating signals for Akt involve the stimulation of receptor and non-receptor tyrosine kinases, and the most potent activator known is the tyrosine phosphatase inhibitor pervanadate, highlighting a possible role for tyrosine phosphorylation in the regulation of the enzyme. In this study we show that activation of Akt by pervanadate or serum is associated with tyrosine phosphorylation of Akt. In addition, in SKOV3 ovarian carcinoma cells that exhibit high basal levels of Akt activity, Akt was tyrosine-phosphorylated in the basal state, and this phosphorylation was further enhanced by both pervanadate and insulin-like growth factor-1. We have used NH(2)-terminal sequencing and phosphate release analysis to directly identify Tyr-474 as the site of tyrosine phosphorylation. Substitution of Tyr-474 with phenylalanine abolished tyrosine phosphorylation of Akt and resulted in up to 55% inhibition of Akt activation, indicating phosphorylation at Tyr-474 is required for full activation of the kinase. Our data identifies a novel regulatory mechanism for this pleiotropic enzyme that may be applicable to the AGC family of protein kinases given the conserved nature of the COOH-terminal hydrophobic motif containing Tyr-474.
Highlights
The protein kinase Akt plays key regulatory roles in a range of physiological processes including glucose metabolism [1, 2], cell survival [3,4,5], proliferation [6, 7], migration [8, 9], and angiogenesis (10 –12)
Understanding the regulation of Akt has been of major interest for elucidating the control of normal cellular physiology as well as malignant transformation
Tyrosine phosphorylation of Akt in pervanadate-treated COS1 cells was abolished by the phosphatidylinositol 3-kinase (PI3K) inhibitor wortmannin, indicating either that the responsible tyrosine kinase is PI3K-dependent or that, like Thr-308 phosphorylation, the tyrosine phosphorylation requires a phosphoinositide-induced conformational change in the kinase [5]
Summary
Materials—The peptide substrate RPRAATF, and carboxyl-terminal peptide from Akt (CRRPHFPQFSYSASSTA) used to raise polyclonal antibodies, were synthesized by Auspep Pty Ltd. The bound antibody was eluted with 7 M urea, 1 M NaCl and 100 mM MOPS, pH 7.0, and dialyzed against phosphatebuffered saline containing 0.01% azide This antibody recognizes the three isoforms of Akt. This antibody recognizes the three isoforms of Akt It was used at 3 g/ml for immunoblotting, and 5 g were used to immunoprecipitate 200 g of protein lysate. In Vitro Kinase Assay—GST-Akt was purified from cell extracts (50 g) using glutathione beads and Akt activity determined by incubation in a total volume of 20 l of 25 mM Tris1⁄7HCl, pH 7.5, 0.5 mM DTT, 0.5 mM benzamidine, 50 M [␥-32P]ATP (5000 cpm/pmol), and 10 mM MgCl2 with 150 M RPRAATF peptide at 30 °C for 10 min. For in vitro reactivation assays GST-⌬PH Akt mutants were purified from extracts of transiently transfected, serum-starved COS1 cells using glutathione beads. The chemical sequence of the phosphopeptides was identified on the same protein sequencer by solid phase sequencing as described previously [23]
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