To be able to assess whether or not the phosphorylation detected working with phospho distinct antibodies represented a degree of effectiveness probably to occur in cells, we performed a lot more comprehensive experiments figuring out the stoichiometry of those phosphorylation occasions in vitro. This showed that CK2 incorporated between 3 and 4 mol phosphate/mol PTEN protein, close to that described previously, although phosphorylation of Ser370 was less efficient than that on the cluster internet sites . GSK3 phosphorylated PTEN poorly with no priming. Even so, after prior phosphorylation with CK2, GSK3 phosphorylated Docetaxel solubility PTEN efficiently upon Thr366. GSK3 did not effectively phosphorylate PTEN if either Thr366 or Ser370 was mutated. Comparison of cellular samples with PTEN phosphorylated to recognized stoichiometry in vitro permitted a crude estimate of your stoichiometry of phosphorylation of these internet sites in cells, suggesting a low amount of phosphorylation of approx. five 10%. Investigating the phosphorylation of PTEN Thr366 by a panel of 37 protein kinases in vitro indicated that other protein kinases exist that will effectively phosphorylate this website experimentally additionally to CK2 and GSK3. We thus chose to address the effects on PTEN phosphorylation of a variety of well characterized smaller molecule protein and lipid kinase inhibitors to determine the extent to which CK2 and GSK3 account for the phosphorylation of those sites in cells.
PTEN phosphorylation upon both Thr366 and Ser370 in cells was inhibited from the CK2 inhibitor DMAT, whereas the GSK3 inhibitors CT99021 and AR A014418 both Estrogen Receptor Pathway inhibited the basal phosphorylation of PTEN on Thr366, but not Ser370.
The specificity of those inhibitors has been tested employing massive panels of protein kinases. Robust suppression of phosphorylation by these inhibitors required extended incubation instances, of several hours or longer, and varied involving cell types, becoming slower when PTEN was expressed in U87MG cells than with endogenous PTEN in NIH 3T3 cells. This suggests that underneath standard situations dephosphorylation of these internet sites is slow. We also addressed other plausible or proposed mechanisms regulating PTEN phosphorylation, including prolinedirected kinase phosphorylation of Thr366, and phosphorylation by ROCK and PI3K dependent feedback phosphorylation. In these experiments, the phosphorylation on Thr366 and Ser370 of wild type PTEN expressed in U87MG cells was not affected by incubation with all the DYRK inhibitor harmine, the MEK inhibitor U0126, the CDK2 inhibitor roscovitine or the ROCK inhibitor Y27632 . The phosphorylation of PTEN on Thr366 and Ser370 was not considerably affected by the PI3K inhibitors LY294002, wortmannin or PI 103 , although a reduction inside the expression amount of PTEN was generally observed, steady together with the previously proposed PI3K feedback regulation of PTEN stability.