Activation Mechanism and Steady State Kinetics of Bruton's Tyrosine Kinase

Abstract

Bruton's tyrosine kinase (BTK) is a member of the Tec nonreceptor 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-NH 2 ) revealed that BTK employs a ternary complex mechanism with K mATP ‫؍‬ 84 ؎ 20 M and K mS1 ‫؍‬ 37 ؎ 8 M. 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 K d is weaker than K m . This investigation of BTK provides the first detailed kinetic characterization of a Tec family kinase. Bruton's tyrosine kinase (BTK)2 is a non-receptor tyrosine kinase that plays an essential role in B cells and other hematopoietic cell types (1-4). The importance of BTK is evident from the fact that mutations in BTK cause X-linked agammaglobulinemia, a human disease that causes defects in both B cell maturation and mature B cell function (5, 6). A BTK point mutation in mice results in a similar, albeit milder, B cell deficiency known as X-linked immunodeficiency (7). The indispensable requirement of BTK for B cell function in humans makes BTK a promising target for treatment of inflammatory diseases that involve inappropriate B cell activation.The BTK-mediated signaling events that link B cell receptor ligation to downstream signaling have been well characterized (8, 9). Upon ligation of the B cell receptor, Src family kinases associated with the receptor, such as Lyn, are the first enzymes activated. This activation results in phosphorylation of tyrosine residues that become docking sites for other kinases including phosphatidylinositol 3-kinase. Once recruited, phosphatidylinositol 3-kinase phosphorylates phosphatidylinositol 4,5-bisphosphate to form phosphatidylinositol 3,4,5-bisphosphate. Formation of phosphatidylinositol 3,4,5-bisphosphate then recruits BTK to the plasma membrane via interaction with its pleckstrin homology domain. Upon recruitment to the membrane, BTK is activated via phosphorylation. BTK proceeds to phosphorylat...

Activation Loop Dynamics Determine the Different Catalytic Efficiencies of B Cell– and T Cell–Specific Tec Kinases

“…3C). The increased activity of the Itk_Btk loop-6 protein came entirely from an increase in k cat (a measure of catalytic rate); the value of k cat for the hyperactive Itk mutant was ~3.5- to 4-fold greater than that of wild-type Itk, and was the same as that of full-length Btk (15, 22). Thus, introduction of six activation segment residues of Btk into full-length Itk activated the mutant Itk similarly to full-length Btk by altering the catalytic rate of the Itk_Btk loop-6 protein, and not by increasing its affinity for its substrates.…”

Identification of an Allosteric Signaling Network within Tec Family Kinases

“…Activity measurements are carried out by monitoring the phosphorylation levels of a peptide substrate in a radioactive assay or by detecting the level of autophosphorylation on the activation loop tyrosine (Y511 in Itk and Y551 in Btk) by western immunoblotting. While phosphorylation of the activation loop tyrosine is achieved by the activity of the Src family kinases Lck and Lyn respectively in vivo , under in vitro conditions Tec kinases autophosphorylate on the activation loop tyrosine[21; 22; 23]. …”

“…Activation of Tec kinases requires the phosphorylation of a conserved tyrosine within the activation loop[12; 21; 23]. Separate high-resolution structures of the Btk kinase domain with Y551 either phosphorylated (active) or unphosphorylated (inactive) have recently been reported[24].…”

Activation Loop Phosphorylation Modulates Bruton’s Tyrosine Kinase (Btk) Kinase Domain Activity

“…Product formation was monitored as a function of time. Using a peptide substrate poly Glu-Tyr (poly EY), we observed a lag phase of ∼40-60 s in Btk product formation for the full-length enzyme (Figure 2A top panel), most likely the result of autophosphorylation on Y 551 (25). ESI-MS analysis indicated that the full-length enzyme was heterogeneously phosphorylated, ranging from 0 to 4 phosphate groups, with 0 and 1 phosphate as the most abundant species ( Figure 3A, top panel).…”