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.