Figure 5. Activation of Btk.

(A) Activation of full-length Btk on membranes. Left panel: Btk (1 μM) autophosphorylates rapidly on lipid vesicles (total lipid concentration, 1 mM) containing 75% DOPC, 20% DOPS and 5% PIP₃. Right panel: end-point autophosphorylation assay of Btk in the presence of lipid vesicles. The molar concentration of DOPC is kept at 75%, and the lipid fractions of PIP₃ is 0%, 2%, 5%, and 10%, with the remaining lipid fraction filled with DOPS. The measurements are performed after 2 min incubation with lipid vesicles in the presence of ATP/Mg²⁺. (B) End-point autophosphorylation assays of Btk in solution in the presence of inositol phosphates. Upper panel: Btk (2 μM) autophosphorylates rapidly in the presence of 100 μM IP₆ in solution, with the results after the first 5 min of incubation shown here. Essentially no phosphorylation is detected in Btk samples incubated with IP₃, IP₄ or I(1,3,4,5,6)P₅, and very low phosphorylation is detected in Btk samples incubated with I(1,2,3,5,6)IP₅ and I(2,3,4,5,6)IP₅ in the same experiment. Lower panel: the assay is repeated with other inositol phosphates at a higher protein concentration (4 μM) and longer incubation time (10 min), and the gel was exposed for a longer time. There is a small amount of degraded Btk in the sample, as judged by the coomassie staining, leading to multiple bands in the western blot shown here and in panel C. (C) Activation of full-length Btk (2 μM) in the presence of IP₆ (100 μM). (D) Activation of full-length Btk (2 μM) with increasing concentrations of IP₆.

DOI: http://dx.doi.org/10.7554/eLife.06074.013

Figure 5—figure supplement 1. Activation of various deletion constructs of Btk.

(A) End-point autophosphorylation assay for full-length Btk, the Src-like module, the SH2-kinase construct and the kinase domain in the presence/absence of IP6. The measurements are carried out after incubating proteins (2 μM) with and without IP₆ (100 μM) for 2 min in the presence of ATP/Mg²⁺. (B) End-point autophosphorylation assay for Btk PH-TH-kinase constructs of various linker lengths in the presence/absence of IP₆ (100 μM). The measurements are carried out after incubating proteins (2 μM) with and without IP₆ (100 μM) for 2 min at the presence of ATP/Mg²⁺. (C) Activation of the PH-TH-kinase construct (2 μM) with a 13-residue linker in the presence and absence of IP₆ (100 μM). (D) Activation of full-length Btk (2 μM) with mutations R133E/Y134E in the presence and absence of IP₆ (100 μM). Arg 133 and Tyr 134 are located at the PH-TH/kinase interface.

DOI: http://dx.doi.org/10.7554/eLife.06074.014