EXAMINATION OF THE DEPHOSPHORYLATION REACTIONS CATALYZED BY PP60(C-SRC) TYROSINE KINASE EXPLORES THE ROLES OF AUTOPHOSPHORYLATION AND SH2 LIGAND-BINDING

pp60(c-src) tyrosine kinase (srcTK) catalyzes the dephosphorylation of phosphotyrosine-containing peptides, including phosphopeptides that b ind with high affinity to the src SH2 domain. The mechanism for these dephosphorylation reactions was investigated. Dephosphorylation was in hibited by a competitive inhibitor for the ATP binding site. In the pr esence of ADP, dephosphorylation of phosphopeptide substrates is prima rily due to the reversal of the kinase reaction. Autoactivated and una ctivated srcTK both catalyzed the reverse of the kinase reaction; howe ver, autoactivated srcTK displayed an increase in k(cat) of approximat ely 4-11-fold relative to unactivated srcTK, depending on the reaction conditions. Autoactivation of srcTK does not affect the K-m's for MgA DP or phosphopeptide (FGE)(3)-pY-(GEF)(2)GD. Unphosphorylated srcTK be comes phosphorylated during the reverse of the kinase reaction upon ac cumulation of free MgaTP. In the presence of MgATP, srcTK also dephosp horylates peptide substrates, by first hydrolyzing MgATP to MgADP. Bin ding of phosphotyrosine peptide ligands to the src SH2 domain stimulat ed the rate of MgATP hydrolysis approximately 2-fold, but had no effec t on the K-m for MgATP. These data suggest that autophosphorylation of tyrosine 419 is not required for nucleotide or peptide binding, or ca talysis involving small peptide substrates. In addition, these results suggest that both the forward and the reverse src tyrosine kinase rea ctions may be important in regulating the intracellular levels of prot ein tyrosine phosphorylation.