Effect of autophosphorylation on the catalytic and regulatory properties of protein tyrosine kinase Src.
The function of autophosphorylation in Src family protein tyrosine kinases is not fully understood. In this paper we compared the catalytic and ligand-binding properties of autophosphorylated and nonautophosphorylated (control) Src. The following are the main differences we found. First, while both forms had the same K(m) for ATP-Mg, autophosphorylated Src had significantly higher K(m) values for the phosphate-accepting substrates, polyE(4)Y, and RCM-lysozyme. The autophosphorylated form also had significantly higher V(max) values than the control. The substrate specificity, as measured by V(max)/K(m) ratio, was altered by autophosphorylation and was dependent on the phosphate-accepting substrate. Second, while autophosphorylation did not affect Src activation by free Mg(2+), Zn(2+), which inhibited Src by competing against an essential Mg(2+) activator, inhibited the control threefold more potently than the autophosphorylated form. Third, autophosphorylation significantly reduced the ability of its SH2 domain to bind phosphotyrosine. Fourth, a Pro-rich Src SH3 domain binding peptide activated the control, but not the autophosphorylated Src even though the apparent binding affinity was not significantly affected by autophosphorylation. These differences indicated that autophosphorylation induced significant and widespread changes in the catalytic and regulatory properties of Src. The implications of these findings relative to Src biological regulation are discussed.
Sun
Gongqin
G
Department of Cell and Molecular Biology, University of Rhode Island, Kingston, Rhode Island 02881, USA. gsun@uri.edu
Ramdas
Latha
L
Wang
Wei
W
Vinci
Jennifer
J
McMurray
John
J
Budde
Raymond J A
RJ
D000081247:CSK Tyrosine-Protein Kinase; D002384:Catalysis; D002712:Chlorides / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008024:Ligands; D008274:Magnesium / Q000737:chemistry; D009113:Muramidase / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D012685:Sepharose / Q000737:chemistry; D012965:Sodium Chloride / Q000494:pharmacology; D013997:Time Factors; D015032:Zinc / Q000737:chemistry; D017967:Zinc Compounds / Q000737:chemistry; D018909:src Homology Domains; D019061:src-Family Kinases
D002712:Chlorides; D008024:Ligands; D010455:Peptides; D011994:Recombinant Proteins; D017967:Zinc Compounds; D019000:Phosphotyrosine; D012965:Sodium Chloride; C016837:zinc chloride; D012685:Sepharose; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; D009113:Muramidase; D008274:Magnesium; D015032:Zinc