Abstract
AKT/PKB is a phosphoinositide-dependent serine/threonine protein kinase that plays a critical role in the signal transduction of receptors. It also serves as an oncogene in the tumorigenesis of cancer cells when aberrantly activated by genetic lesions of the PTEN tumor suppressor, phosphatidylinositol 3-kinase, and receptor tyrosine kinase overexpression. Here we have characterized and compared kinetic mechanisms of the three AKT isoforms. Initial velocity studies revealed that all AKT isozymes follow the sequential kinetic mechanism by which an enzyme-substrate ternary complex forms before the product release. The empirically derived kinetic parameters are apparently different among the isoforms. AKT2 showed the highest Km value for ATP, and AKT3 showed the highest kcat value. The patterns of product inhibition of AKT1, AKT2, and AKT3 by ADP were all consistent with an ordered substrate addition mechanism with ATP binding to the enzymes prior to the peptide substrate. Further analysis of steady state kinetics of AKT1 in the presence of dead-end inhibitors supported the finding and suggested that the AKT family of kinases catalyzes reactions via an Ordered Bi Bi sequential mechanism with ATP binding to the enzyme prior to peptide substrate and ADP being released after the phosphopeptide product. These results suggest that ATP is an initiating factor for the catalysis of AKT enzymes and may play a role in the regulation AKT enzyme activity in cells.
Highlights
(AKT1) in the C-terminal hydrophobic motif [14]
Activation of Akt is triggered by the interaction between the lipid products of PI 3-kinase and the PH domain of AKT, which induces a conformational change in AKT exposing the activation segment of the kinase domain. This serves to recruit the 3-phosphoinositide-dependent protein kinase PDK1 to the proximity of the activation segment allowing the phosphorylation of Thr-308 (Thr-309 for AKT2 and Thr-305 for AKT3) [15,16,17,18, 20]
One possible explanation for the isoformspecific functions could be the differential tissue expression pattern of the AKT isoforms because tissues types where the major defect resides in the AKT1, AKT2, and AKT3 knock-out mice somewhat correlate with the expression of a particular AKT isoform
Summary
Materials—ATP, ADP, and AMP-PCP were purchased from Sigma. [␥-33P]ATP (3000 Ci/mmol) was purchased from PerkinElmer Life Sciences. Insect cell paste (60 –100 g) was resuspended in 5 ml of lysis buffer per g (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM imidazole, 10% glycerol, 5 mM DTT, 1ϫ Complete (Roche Applied Science)) and lysed by nitrogen cavitation in a Parr bomb. ATP and MgCl2 were added to 1 and 10 mM, respectively, and the final reaction volume was adjusted to 8 ml with activation buffer. Tion mixture contained 20 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 1 mM DTT, 1 mM EGTA, 1 mM Na3VO4, 0.1% bovine serum albumin (kinase reaction buffer), with the addition of activated AKT. The reaction mixtures were incubated at room temperature for 10 min and stopped by adding 100 l of 5% phosphoric acid Under these conditions, all initial velocity reactions utilized less than 10% of the substrates. The experimental details and data analysis are as described under “Experimental Procedures.”
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