Phosphorylation of PTP1B at Ser50 by Akt Impairs Its Ability to Dephosphorylate the Insulin Receptor

LV Ravichandran, H Chen, Y Li… - Molecular …, 2001 - academic.oup.com
LV Ravichandran, H Chen, Y Li, MJ Quon
Molecular endocrinology, 2001academic.oup.com
PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by
dephosphorylating the insulin receptor. Akt is a ser/thr kinase effector of insulin signaling
that phosphorylates substrates at the consensus motif RXRXXS/T. Interestingly, PTP1B
contains this motif (RYRDVS50), and wild-type PTP1B (but not mutants with substitutions for
Ser50) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a
substrate for Akt in intact cells, NIH-3T3IR cells transfected with either wild-type PTP1B or …
Abstract
PTP1B is a protein tyrosine phosphatase that negatively regulates insulin sensitivity by dephosphorylating the insulin receptor. Akt is a ser/thr kinase effector of insulin signaling that phosphorylates substrates at the consensus motif RXRXXS/T. Interestingly, PTP1B contains this motif (RYRDVS50), and wild-type PTP1B (but not mutants with substitutions for Ser50) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a substrate for Akt in intact cells, NIH-3T3IR cells transfected with either wild-type PTP1B or PTP1B-S50A were labeled with[ 32P]-orthophosphate. Insulin stimulation caused a significant increase in phosphorylation of wild-type PTP1B that could be blocked by pretreatment of cells with wortmannin or cotransfection of a dominant inhibitory Akt mutant. Similar results were observed with endogenous PTP1B in untransfected HepG2 cells. Cotransfection of constitutively active Akt caused robust phosphorylation of wild-type PTP1B both in the absence and presence of insulin. By contrast, PTP1B-S50A did not undergo phosphorylation in response to insulin. We tested the functional significance of phosphorylation at Ser50 by evaluating insulin receptor autophosphorylation in transfected Cos-7 cells. Insulin treatment caused robust receptor autophosphorylation that could be substantially reduced by coexpression of wild-type PTP1B. Similar results were obtained with coexpression of PTP1B-S50A. However, under the same conditions, PTP1B-S50D had an impaired ability to dephosphorylate the insulin receptor. Moreover, cotransfection of constitutively active Akt significantly inhibited the ability of wild-type PTP1B, but not PTP1B-S50A, to dephosphorylate the insulin receptor. We conclude that PTP1B is a novel substrate for Akt and that phosphorylation of PTP1B by Akt at Ser50 may negatively modulate its phosphatase activity creating a positive feedback mechanism for insulin signaling.
Oxford University Press