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

Abstract

Itk and Btk are nonreceptor tyrosine kinases of the Tec family that signal downstream of the T cell receptor (TCR) and B cell receptor (BCR), respectively. Despite their high sequence similarity and related signaling roles, Btk is a substantially more active kinase than Itk. We showed that substitution of six of the 619 amino acid residues of Itk with those of Btk was sufficient to completely switch the activities of Itk and Btk. The substitutions responsible for the swap in activity are all localized to the activation segment of the kinase domain. Nuclear magnetic resonance and hydrogen-deuterium exchange mass spectrometry analyses revealed that Itk and Btk had distinct protein dynamics in this region, which could explain the observed differences in catalytic efficiency between these kinases. Introducing Itk with enhanced activity into T cells led to enhanced and prolonged TCR signaling compared to that in cells with wild-type Itk. These findings imply that evolutionary pressures have led to Tec kinases having distinct enzymatic properties depending on the cellular context. We suggest that the weaker catalytic activities observed for T cell–specific kinases is one mechanism to regulate cellular activation and prevent aberrant immune responses.

The Accessory Factor Nef Links HIV-1 to Tec/Btk Kinases in an Src Homology 3 Domain-dependent Manner

“…As shown in Fig. 10A, Itk was not detectably autophosphorylated when expressed alone in 293T cells, consistent with low intrinsic kinase activity as reported elsewhere (46). However, co-expression of Itk with Nef resulted in a dramatic increase in Itk phosphotyrosine content, consistent with Nefdependent enhancement of kinase activity.…”

An Autoinhibitory Role for the Pleckstrin Homology Domain of Interleukin-2-Inducible Tyrosine Kinase and Its Interplay with Canonical Phospholipid Recognition

“…The catalytically inactive full length ITK (K390R) and ITK SH3-SH2-Kinase (K390R) are used in this assay to ensure that phosphorylation on ITK Y511 is solely due to LCK activity and not due to intrinsic ITK activity. Thus, the extent to which ITK Y511 is phosphorylated by exogenous LCK reveals the accessibility of the ITK activation loop under different conditions 25 . Both full length ITK (K390R) and the ITK SH3-SH2-Kinase (K390R) fragment are subjected to phosphorylation by active LCK in the presence and absence of PIP 3 .…”

A Conserved Isoleucine Maintains the Inactive State of Bruton's Tyrosine Kinase

“…2a). Activation loop phosphorylation is commonly used as a qualitative indicator of kinase activity (26, 27). Western blot analysis indicates that the triple mutant (Btk IEE/LPA) is more active than wild type Btk, consistent with analysis of the Btk simulation data suggesting that I432, E439 and E441 contribute to the destabilization of the active Btk conformation.…”

“…Overall, NMR linewidths are broader for the Btk I432L mutant than wild type Btk suggesting that the activated mutant is more dynamic. Complete backbone resonance assignments for the Btk kinase domain are not available and so we have pursued selective isotopic labeling to obtain chemical shift assignments for the phenylalanine backbone amide resonances in the Btk kinase domain (27, 28). …”

“…Having previously assigned the backbone amide of F540 (27), we examined its resonance in 1 H- 15 N TROSY HSQC spectra of the Btk wild type and I432L mutant kinase domains. The location of F540 within the conserved DFG motif places it close in space to the αC helix (Figs.…”