TY - JOUR
T1 - Src regulated by C-terminal phosphorylation is monomeric
AU - Weijland, Albert
AU - Williams, John C.
AU - Neubauer, Gitte
AU - Courtneidge, Sara A.
AU - Wierenga, Rik K.
AU - Superti-Furga, Giulio
PY - 1997/4/15
Y1 - 1997/4/15
N2 - The activity of the c-Src protein tyrosine kinase is regulated by phosphorylation of a tyrosine residue (Tyr-527) in the C-terminal tail of the molecule. Phosphorylation of Tyr-527 promotes association of the tail with the SH2 domain and a concomitant reduction of the enzymatic activity of Src. We asked the question whether regulation by C-terminal phosphorylation was accompanied by a change in the quaternary structure 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 the unique domain from Schizosaccharomyces pombe cells. The cells were engineered to express Src along with Csk, a protein kinase able to phosphorylate 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 phosphorylated peptide able to bind the SH2 domain with high affinity. Using gel filtration chromatography, dynamic light scattering, and ultracentrifugation, we found that the regulated form of Src was a monomer. We have obtained crystals diffracting to 2.4 Å with space group P212121 and one molecule per asymmetric unit, in agreement with the monomeric state. These results indicate that the structural rearrangements of regulated Src are of an intramolecular nature.
AB - The activity of the c-Src protein tyrosine kinase is regulated by phosphorylation of a tyrosine residue (Tyr-527) in the C-terminal tail of the molecule. Phosphorylation of Tyr-527 promotes association of the tail with the SH2 domain and a concomitant reduction of the enzymatic activity of Src. We asked the question whether regulation by C-terminal phosphorylation was accompanied by a change in the quaternary structure 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 the unique domain from Schizosaccharomyces pombe cells. The cells were engineered to express Src along with Csk, a protein kinase able to phosphorylate 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 phosphorylated peptide able to bind the SH2 domain with high affinity. Using gel filtration chromatography, dynamic light scattering, and ultracentrifugation, we found that the regulated form of Src was a monomer. We have obtained crystals diffracting to 2.4 Å with space group P212121 and one molecule per asymmetric unit, in agreement with the monomeric state. These results indicate that the structural rearrangements of regulated Src are of an intramolecular nature.
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U2 - 10.1073/pnas.94.8.3590
DO - 10.1073/pnas.94.8.3590
M3 - Article
C2 - 9108021
AN - SCOPUS:0030891301
SN - 0027-8424
VL - 94
SP - 3590
EP - 3595
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 8
ER -