AUTOPHOSPHORYLATION INDUCES AUTOACTIVATION AND A DECREASE IN THE SRC HOMOLOGY-2 DOMAIN ACCESSIBILITY OF THE LYN PROTEIN-KINASE

Lyn is a member of the Src family of protein-tyrosine kinases that can readily undergo autophosphorylation in vitro, The site of autophospho rylation is Tyr(397) which corresponds to the consensus autophosphoryl ation site of other Src family tyrosine kinases, The rate of autophosp horylation is concentration-dependent, indicating that the reaction fo llows an intermolecular mechanism, Autophosphorylation results in a 17 -fold increase in protein-tyrosine kinase activity. Kinetic analysis d emonstrates that phosphorylation of a substrate peptide by Lyn followi ng autophosphorylation occurs with a 63-fold decrease in K-m but no si gnificant change in V-max suggesting that autophosphorylation relieves the conformational constraint that prevents binding of the substrate peptide to the active site of the kinase, Using a phosphotyrosine-cont aining peptide (pYEEI) that has previously been shown to bind to the S rc homology 2 (SH2) domain of Src family tyrosine kinases with high af finity, we found that autophosphorylation results in a significant dec rease in accessibility of the Lyn SH2 domain, indicating that conforma tional changes in the protein kinase domain induced by autophosphoryla tion can be propagated to the SH2 domain, Our study suggests that auto phosphorylation plays an important role in regulating Lyn by modulatin g both its kinase activity and its interaction with other phosphotyros ine-containing molecules.