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. 1997 Oct 14;94(21):11227–11232. doi: 10.1073/pnas.94.21.11227

Figure 4.

Figure 4

Requirement for the PH of ITK for Src-induced membrane localization and activation. (A) Src induces membrane localization of ITK. COS-7 cells were transfected with HA-tagged ITK in the presence or absence of Src. ITK was then immunoprecipitated from cytosolic and soluble membrane fraction and analyzed for phosphotyrosine content. Lanes: 1, cytosolic fraction/ITK alone; 2, cytosolic fraction/ITK plus Src; 3, membrane fraction/ITK alone; 4, membrane fraction/ITK plus Src. (B) The PH is required for Src-induced membrane association of ITK. COS-7 cells were transfected with HA-tagged ΔPH ITK in the presence or absence of Src. ΔPH ITK was immunoprecipitated from each fraction described above and analyzed for phosphotyrosine content. Lanes: 1, cytosolic fraction/ΔPH ITK alone; 2, cytosolic fraction/ΔPH ITK plus Src; 3, membrane fraction/ΔPH ITK alone; 4, membrane fraction/ΔPH ITK plus Src. Top was probed with anti-phosphotyrosine; Middle with anti-HA. Bottom shows equal amounts of protein from each fraction probed with anti-Src. Arrows point to ITK and Src, respectively. (C) Binding of the PH of ITK to inositol phosphates. Purified glutathione S-transferase-PH proteins were incubated with the indicated inositol phosphates as described in Materials and Methods. I 1-P, inositol 1-monophosphate; I 1,3,4-P; inositol 1,3,4-triphosphate; I 1,4,5-P, inositol 1,4,5-triphosphate; I 1,3,4,5-P, inositol 1,3,4,5-tetraphosphate; I 1,2,3,4,5-P, inositol 1,2,3,4,5-hexaphosphate. (D) Requirement for the PH of ITK for Src-induced activation. COS-7 cells were transfected with the HA-tagged ΔPH mutant of ITK in the presence or absence of c-Src. ITK immunoprecipitates were then analyzed for enzymatic activity (Fig. 2A) and phosphotyrosine content. Lanes: 1 and 3, ΔPH ITK without c-Src; 2 and 4, ΔPH ITK with c-Src. Lanes 3 and 4 were from cells treated with Ly294002. Probes: anti-phosphotyrosine (Top); anti-HA antibody (Middle); and (Bottom) anti-Src antibodies on whole cell lysates. Arrows point to the different mutants of ITK or c-Src. (E) Membrane bound ΔPH ITK recovers ability to be activated by Src. COS-7 cells were transfected with either Kit/ΔPH ITK alone or in the presence of c-Src. ITK was immunoprecipitated and analyzed for phosphotyrosine content. Lanes: 1 and 3, Kit/ΔPH ITK alone; 2 and 4, Kit/ΔPH ITK plus c-Src. Lanes 3 and 4 were from cells treated with Ly294002. Probes: (Top) anti-phosphotyrosine, (Middle) anti-ITK; and (Bottom) anti-src antibodies on whole cell lysates. Arrows indicate ITK or c-Src.