Activation mechanism and steady state kinetics of Bruton's tyrosine kinase.
Bruton's tyrosine kinase (BTK) is a member of the Tec non-receptor tyrosine kinase family that is involved in regulating B cell proliferation. To better understand the enzymatic mechanism of the Tec family of kinases, the kinetics of BTK substrate phosphorylation were characterized using a radioactive enzyme assay. We first examined whether autophosphorylation regulates BTK activity. Western blotting with a phosphospecific antibody revealed that BTK rapidly autophosphorylates at Tyr(551) within its activation loop in vitro. Examination of a Y551F BTK mutant indicated that phosphorylation of Tyr(551) causes a 10-fold increase in BTK activity. We then proceeded to characterize the steady state kinetic mechanism of BTK. Varying the concentrations of ATP and S1 peptide (biotin-Aca-AAAEEIY-GEI-NH2) revealed that BTK employs a ternary complex mechanism with KmATP = 84 +/- 20 microM and KmS1 = 37 +/- 8 microM. Inhibition studies were also performed to examine the order of substrate binding. The inhibitors ADP and staurosporine were both found to be competitive with ATP and non-competitive with S1, indicating binding of ATP and S1 to BTK is either random or ordered with ATP binding first. Negative cooperativity was also found between the S1 and ATP binding sites. Unlike ATP site inhibitors, substrate analog inhibitors did not inhibit BTK at concentrations less than 1 mm, suggesting that BTK may employ a "substrate clamping" type of kinetic mechanism whereby the substrate Kd is weaker than Km. This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase.
Dinh
Marie
M
Department of Biochemical Pharmacology, Roche Palo Alto LLC, CA 94304, USA.
Grunberger
Dorit
D
Ho
Hoangdung
H
Tsing
Stan Y
SY
Shaw
David
D
Lee
Simon
S
Barnett
Jim
J
Hill
Ronald J
RJ
Swinney
David C
DC
Bradshaw
J Michael
JM
D000244:Adenosine Diphosphate / Q000737:chemistry; D000255:Adenosine Triphosphate / Q000737:chemistry; D000077329:Agammaglobulinaemia Tyrosine Kinase; D001665:Binding Sites; D001667:Binding, Competitive; D004789:Enzyme Activation; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D019311:Staurosporine / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity; D014443:Tyrosine / Q000737:chemistry
D010455:Peptides; D014443:Tyrosine; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011505:Protein-Tyrosine Kinases; D000077329:Agammaglobulinaemia Tyrosine Kinase; C000625949:BTK protein, human; D019311:Staurosporine