The Protein-tyrosine-phosphatase SHP2 is phosphorylated on serine residues 576 and 591 by protein kinase C isoforms alpha, beta 1, beta 2, and eta.
To study whether protein kinase C (PKC) isoforms can interact with protein-tyrosine-phosphatases (PTPs) which are connected to the insulin signaling pathway, we co-overexpressed PKC isoforms together with insulin receptor, docking proteins, and the PTPs SHP1 and SHP2 in human embryonic kidney (HEK) 293 cells. After phorbol ester induced activation of PKC isoforms alpha, beta 1, beta 2, and eta, we could show a defined gel mobility shift of SHP2, indicating phosphorylation on serine/threonine residues. This phosphorylation was not dependent on insulin receptor or insulin receptor substrate-1 (IRS-1) overexpression and did not occur for the closely related phosphatase SHP1. Furthermore, PKC phosphorylation of SHP2 was completely blocked by the PKC inhibitor bisindolylmaleimide and was not detectable when SHP2 was co-overexpressed with kinase negative mutants of PKC beta 1 and -beta 2. The phosphorylation also occurred on endogenous SHP2 in Chinese hamster ovary (CHO) cells stably overexpressing PKC beta 2. Using point mutants of SHP2, we identified serine residues 576 and 591 as phosphorylation sites for PKC. However, no change of phosphatase activity by TPA treatment was detected in an in vitro assay. In summary, SHP2 is phosphorylated on serine residues 576 and 591 by PKC isoforms alpha, beta 1, beta 2, and eta.
Strack
Volker
V
Medical Clinic, Department IV, Eberhard-Karls-University Tübingen, Otfried-Müller-Strasse 10, D-72076 Tübingen, Germany.
Krützfeldt
Jan
J
Kellerer
Monika
M
Ullrich
Axel
A
Lammers
Reiner
R
Häring
Hans-Ulrich
HU
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D015801:Helminth Proteins / Q000737:chemistry*; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry*; D009154:Mutation; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000737:chemistry*; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha; D012694:Serine / Q000737:chemistry*
D018076:DNA, Complementary; D015801:Helminth Proteins; D007527:Isoenzymes; D020033:Protein Isoforms; C095048:microfilarial sheath protein, Helminth; D012694:Serine; C089212:protein kinase D; C496628:PRKCA protein, human; D011493:Protein Kinase C; D064546:Protein Kinase C beta; D051571:Protein Kinase C-alpha