title: Phosphotyrosyl turnover in insulin signaling. Characterization of two membrane-bound pp15 protein tyrosine phosphatases from 3T3-L1 adipocytes.
abstract: It was shown previously that 422 (aP2) protein, a 15-kDa fatty acid binding protein, is phosphorylated on Tyr19 both in vitro by the insulin receptor tyrosine kinase and in intact 3T3-L1 adipocytes treated with insulin and phenylarsine oxide (PAO). Phospho-422(aP2) protein (pp15) accumulates in cells treated with insulin and PAO because the arsenical blocks turnover of the phosphoryl group of pp15. These findings suggest that a PAO-sensitive enzyme mediates turnover of the pp15 tyrosine phosphoryl group. We have purified and characterized two membrane protein tyrosine phosphatases (PTPases) from 3T3-L1 adipocytes that catalyze hydrolysis of phospho-Tyr19 of authentic pp15. These enzymes, designated PTPases HA1 and HA2, were purified approximately 20,000-fold and approximately 15,000-fold, respectively, and shown to differ markedly in their sensitivity to both vanadate and phosphotyrosine. Both enzymes are inhibited by PAO and accordingly can be labeled with 4-[125I]iodo-PAO. By this method, it was demonstrated that PTPases HA1 and HA2 have molecular masses of approximately 60 kDa and approximately 38 kDa, respectively. Both enzymes exhibit substrate preference for pp15 when compared with other phosphotyrosine-containing protein substrates. Proteins containing phosphoserine and phosphothreonine do not serve as substrates for the enzymes. The pp15 PTPase HA2 is expressed both in 3T3-L1 preadipocytes and adipocytes, whereas pp15 PTPase HA1 is expressed only in 3T3-L1 adipocytes.
authors.affiliation: Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors.forename: K
authors.initials: K
authors.lastname: Liao
authors.forename: R D
authors.initials: RD
authors.lastname: Hoffman
authors.forename: M D
authors.initials: MD
authors.lastname: Lane
mesh_terms: D000273:Adipose Tissue / Q000166:cytology / Q000201:enzymology*; D001152:Arsenicals / Q000737:chemistry; D002478:Cells, Cultured; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000378:metabolism*; D007700:Kinetics; D008565:Membrane Proteins / Q000378:metabolism; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases; D013379:Substrate Specificity
chemical_list: D001152:Arsenicals; D007328:Insulin; D008565:Membrane Proteins; C029341:oxophenylarsine; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases