Rane with a fusion protein consisting of Akt1 and the signal was associated with constitutive myristoylation occurred T308 phosphorylation in H9c2 myoblasts, in a culture medium lacking exogenous growth factors. Add an allosteric inhibitor of Akt in these cells leads to dephosphorylation MyrAkt1 within 30 minutes JNJ-7706621 and dephosphorylation was inhibited by phosphatase inhibitors, S Okadaic acid That or calyculin. It also prevents ATP-competitive inhibitor of Akt, a 443 654, VIII-inhibitor-induced Akt dephosphorylation more effectively as phosphatase inhibitors. To determine whether a 443 654 also inhibits the dephosphorylation of wild-type Akt1 were H9c2 cells by insulin for 20 min in order to induce the endogenous Akt phosphorylation stimulated.
To perform act dephosphorylation, the cells were then incubated in a medium without growth factor with and without the PDK1 inhibitor, UCN01 or the PI 3-kinase inhibitor, wortmannin. Under these conditions, a delay Gerung 443 654 of both T308 and S473 act dephoshorylation pages. We have also observed that under the terms of the growth factor starved and 443 654 treatment Flavopiridol CDK inhibitor alone only slightly Akt1 phosphorylation, a leistungsf other 443 654 Hige synergy with insulin to induce hyperphosphorylation induced Akt1 at T308. These results are consistent with the hypothesis that Akt induces hyperphosphorylation by the inhibition of Akt 443 654 dephosphorylation. Three-dimensional structures of the kinase inhibitor complexed with Akt VIII show that the inhibitor VIII may access from ATP to the kinase-Dom Ne act of Restrict Nken stabilizing interactions between the PH-Dom And a kinase-ne Cathedral sharing plans.
In contrast, A 443 654, as ATP-competitive inhibitor, binding to the ATP-binding pocket with high affinity t. To test whether regulates the occupation of the ATP-binding pocket act T308 phosphorylation, we mutated the ATP binding of lysine residues in Akt1 Evodiamine Myr to methionine. As a contr Them, we also generated mutants that have abolished the target of phosphorylation in the activation loop. On transfection into H9c2 cells, this mutant K179M MyrAkt1 station Safe state, the phosphorylation of T308 was reduced to a level Similar to that of the T308A mutant. The confocal microscopy and membrane fractionation best Firmed that the K179M mutation Changed nothing in the subcellular Ren localization Akt1 Myr.
Together, these pharmacological and genetic data of the model is that the occupation of the acceptor site of ATP in the kinase-Akt affects the kinetics of dephosphorylation of T308. Acceptor site occupancy of ATP by ATP and its analogues resistant T308 dephosphorylation in vitro. Next we determined whether the regulated occupation of the acceptor site of ATP dephosphorylation in cell extracts T308. To this end, constitutive Akt1 Myr in HeLa cells phosphorylated by rapid freezing with dry ice / subsequently Extractant was expressed. On further incubation of cell extracts at 30 MyrAkt1 was within 15 minutes and two and calyculin A dephosphorylates inhibitor of AKT 443 654 independent Ngig blocked T308 dephosphorylation. But in contrast to calyculin, 443 654 A inhibits Akt1 phosphorylation but not dephosphorylation of GSK, PRAS40 or ERK1. Thus, in cell extracts without kinase activity t due to the presence of EDTA, a 443,654 specifically inhibited dephosphorylation MyrAkt1. This effect extends to a constitutively phosphorylated Akt 443 654 WT by Vana