Identification, characterization and purification to near-homogeneity of a novel 67 kDa phosphotyrosyl protein phosphatase associated with pig lung annexin extract.
During the purification of annexin VI from pig lung, we previously reported the isolation of another 67 kDa protein (protein 67E) differing from the former by immunological reactivity, amino acid composition, inability to interact with anionic phospholipids in the presence of Ca2+ and inability to inhibit phospholipase A2 [Fauvel, Vicendo, Roques, Ragab-Thomas, Granier, Vilgrain, Chambaz, Rochat, Chap & Douste-Blazy (1987) FEBS Lett. 221, 397-402]. Attempts to phosphorylate protein 67E by the protein tyrosine kinase of epidermal-growth-factor receptor revealed a dramatic inhibition of receptor autophosphorylation, which was also observed with insulin receptor. This inhibitory effect was found to be supported by a phosphatase active towards p-nitrophenyl phosphate, phosphotyrosine, [32P]phosphotyrosyl histones and [32P]phosphotyrosyl poly(Glu,Tyr), but inactive towards phosphoserine, phosphothreonine and [32P]phosphoseryl histones. Although not purified to complete homogeneity, the enzyme was purified 273-fold over EGTA extracts from pig lung and corresponded to a monomeric protein displaying an apparent molecular mass of 67 kDa. With [32P]phosphotyrosyl poly(Glu,Tyr) as substrate, the purified enzyme displayed Km and Vmax. values of 10 microM and 1.93 mumol/min per mg respectively, which compare reasonably well with other recently described phosphotyrosyl protein phosphatases. From these data and from its sensitivity to various inhibitors, it is concluded that protein fraction 67E contains a novel phosphotyrosyl protein phosphatase, the association of which with annexin extract might offer a clue to the understanding of its possible targeting to membrane substrates.
Vicendo
P
P
I.N.S.E.R.M, Unité 326. Hôpital Purpan, Toulouse, France.
Fauvel
J
J
Ragab-Thomas
J M
JM
Chap
H
H
D000818:Animals; D017317:Annexin A6; D002135:Calcium-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008168:Lung / Q000737:chemistry*; D008970:Molecular Weight; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D017027:Protein Tyrosine Phosphatases; D011972:Receptor, Insulin / Q000378:metabolism; D013379:Substrate Specificity; D013552:Swine
D017317:Annexin A6; D002135:Calcium-Binding Proteins; D010750:Phosphoproteins; D066246:ErbB Receptors; D011972:Receptor, Insulin; D010749:Phosphoprotein Phosphatases; D017027:Protein Tyrosine Phosphatases