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Phosphorylation of PTP1B at Ser(50) by Akt impairs its ability to dephosphorylate the insulin receptor.

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 (RYRDVS(50)), and wild-type PTP1B (but not mutants with substitutions for Ser(50)) was significantly phosphorylated by Akt in vitro. To determine whether PTP1B is a substrate for Akt in intact cells, NIH-3T3(IR) cells transfected with either wild-type PTP1B or PTP1B-S50A were labeled with [(32)P]-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 Ser(50) 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 Ser(50) may negatively modulate its phosphatase activity creating a positive feedback mechanism for insulin signaling.

Pubmed ID: 11579209

Authors

  • Ravichandran LV
  • Chen H
  • Li Y
  • Quon MJ

Journal

Molecular endocrinology (Baltimore, Md.)

Publication Data

October 1, 2001

Associated Grants

None

Mesh Terms

  • 3T3 Cells
  • Amino Acid Sequence
  • Animals
  • COS Cells
  • Consensus Sequence
  • Feedback
  • Humans
  • Insulin
  • Mice
  • Molecular Sequence Data
  • Mutation
  • Phosphorylation
  • Phosphoserine
  • Plasmids
  • Protein Tyrosine Phosphatase, Non-Receptor Type 1
  • Protein Tyrosine Phosphatases
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-akt
  • Receptor, Insulin
  • Signal Transduction
  • Transfection