Identification of an Allosteric Signaling Network within Tec Family Kinases

Abstract

The Tec family kinases are tyrosine kinases that function primarily in hematopoietic cells. The catalytic activity of the Tec kinases is positively influenced by the regulatory domains outside of the kinase domain. The current lack of a full-length Tec kinase structure leaves a void in our understanding of how these positive regulatory signals are transmitted to the kinase domain. Recently, a conserved structure within kinases, the ‘regulatory spine’, has been identified that assembles and disassembles as a kinase switches between its active and inactive states. Here we define the residues that comprise the regulatory spine within Tec kinases. Compared to previously characterized systems, the Tec kinases contain an extended regulatory spine that includes a conserved methionine within the C-helix and a conserved tryptophan within the SH2-kinase linker of Tec kinases. This extended regulatory spine forms a conduit for transmitting the presence of the regulatory domains of Tec kinases to the catalytic domain. We further show that mutation of the gatekeeper residue at the edge of the regulatory spine stabilizes the regulatory spine resulting in a constitutively active kinase domain. Importantly, the regulatory spine is preassembled in this gatekeeper mutant rendering phosphorylation on the activation loop unnecessary for its activity. Moreover, we show that the disruption of the conserved electrostatic interaction between Btk R544 on the activation loop and Btk E445 on the C-helix also aids in the assembly of the regulatory spine. Thus, the extended regulatory spine is a key structure that is critical for maintaining the activity of Tec kinases.

Bruton’s Tyrosine Kinase Mediates FcγRIIa/Toll-Like Receptor–4 Receptor Crosstalk in Human Neutrophils

“…These observations agree with the data presented in Figures 3B and 3C. We note that the anti-Btk antibody used in these experiments (Western blotting) recognizes a kinase domain of Btk, thereby detecting Btk in its active state (36).…”

“…However, they are significantly increased in neutrophils treated with anti-IL-8:IL-8 immune complexes, and in cells that were "primed" with LPS and activated by the complexes ( Figure 3A, upper blot). Notably, the anti-Btk antibody used in these experiments, as well as in immunofluorescence staining ( Figure 3C, bottom), recognizes a kinase domain of Btk, and thereby detects Btk in its activated state (36). On the other hand, when an antibody against total Btk is used, no difference in Btk concentrations is evident ( Figure 3A, middle blot).…”

“…Similar results were obtained for Btk (Figure 5B), in agreement with our observations that FcgRIIa mediates the activation of Btk in neutrophils "primed" with LPS and stimulated with anti-IL-8:IL-8 immune complexes. We note that the anti-Btk antibody used in these experiments (Western blotting) recognizes a kinase domain of Btk, thereby detecting Btk in its active state (36).…”

“…The transfection of these cells with cDNA for Btk resulted in an increase in the basal expression of Btk (not shown), and a substantial increase in the amount of Btk in cells treated with the complexes (Figures 6A and 6B). The data presented in Figures 6A-6C were obtained either antitotal Btk antibody ( Figure 6A) or the antibody that recognizes a kinase domain of Btk, and therefore detects Btk in its active state ( Figures 6A-6C) (36).…”

Molecular Characteristics of CTA056, a Novel Interleukin-2-Inducible T-Cell Kinase Inhibitor that Selectively Targets Malignant T Cells and Modulates Oncomirs

“…We attribute this to the differential specificities of these inhibitors toward other kinases or toward different conformational isoforms of Itk (e.g., monomer versus multimer, phosphorylated versus unphosphorylated, etc. ), which may engage different substrates (Dombroski et al, 2005;Qi and August, 2009;Joseph et al, 2010). As discussed above, our inhibitor also inhibits Src kinase, albeit with less potency, which may contribute to the observed apoptosis-inducing effects.…”