SRC REGULATED BY C-TERMINAL PHOSPHORYLATION IS MONOMERIC

The activity of the c-Src protein tyrosine kinase is regulated by phos phorylation of a tyrosine residue (Tyr-527) in the C-terminal tail of the molecule. Phosphorylation of Tyr-527 promotes association of the t ail with the SH2 domain and a concomitant reduction of the enzymatic a ctivity of Src. We asked the question whether regulation by C-terminal phosphorylation was accompanied by a change in the quaternary structu re of the enzyme or if it occurred within a monomeric form of Src. For this purpose we purified to homogeneity a chicken Src form lacking th e unique domain from Schizosaccharomyces pombe cells. The cells were e ngineered to express Src along with Csk, a protein kinase able to phos phorylate Tyr-527 efficiently. Mass spectrometric analysis showed that purified Src was homogeneously phosphorylated at Tyr-527. The enzyme was in the regulated form, because it could be activated by a phosphor ylated peptide able to bind the SH2 domain with high affinity. Using g el filtration chromatography, dynamic light scattering, and ultracentr ifugation, we found that the regulated form of Src was a monomer. We h ave obtained crystals diffracting to 2.4 Angstrom with space group P2( 1)2(1)2(1) and one molecule per asymmetric unit, in agreement with the monomeric state. These results indicate that the structural rearrange ments of regulated Src are of an intramolecular nature.