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. 1997 Oct 14;94(21):11227-32.
doi: 10.1073/pnas.94.21.11227.

Src-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the Pleckstrin homology domain of inducible T cell kinase

A August  1 , A Sadra, B Dupont, H Hanafusa
Affiliations
Free PMC article

Src-induced activation of inducible T cell kinase (ITK) requires phosphatidylinositol 3-kinase activity and the Pleckstrin homology domain of inducible T cell kinase

A August et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

The Tec family of tyrosine kinases are involved in signals emanating from cytokine receptors, antigen receptors, and other lymphoid cell surface receptors. One family member, ITK (inducible T cell kinase), is involved in T cell activation and can be activated by the T cell receptor and the CD28 cell surface receptor. This stimulation of tyrosine phosphorylation and activation of ITK can be mimicked by the Src family kinase Lck. We have explored the mechanism of this requirement for Src family kinases in the activation of ITK. We found that coexpression of ITK and Src results in increased membrane association, tyrosine phosphorylation and activation of ITK, which could be blocked by inhibitors of the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase) as well as overexpression of the p85 subunit of PI 3-kinase. Removal of the Pleckstrin homology domain (PH) of ITK resulted in a kinase that could no longer be induced to localize to the membrane or be activated by Src. The PH of ITK was also able to bind inositol phosphates phosphorylated at the D3 position. Membrane targeting of ITK without the PH recovered its ability to be activated by Src. These results suggest that ITK can be activated by a combination of Src and PI 3-kinase.

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Figures

Figure 1
Figure 1
Structure of the different ITKs used in this study. (A) HAΔPH ITK: HA-tagged PH-deleted ITK; Kit/ΔPH ITK: murine c-Kit/HAΔPH ITK fusion. (B) Expression and kinase activity of ITK and mutants. Immunoprecipitates of ITK or mutants from transfected COS-7 cells were analyzed for autokinase activity (Top). Lanes: 1, ITK (wild-type); 2, ΔPH ITK; 3, Kit/ΔPH ITK. (Bottom) Probed with anti-HA. Arrows point to wild-type ITK or the mutants. (C) Tyrosine phosphorylation of ITK in the presence of Src and inhibition by Ly294002. COS-7 cells transfected with untagged wild-type ITK (except for lanes 5 and 6, where HA-tagged wild-type ITK was used) in the presence or absence of wild-type or kinase-inactive (K295M) c-Src as indicated. ITK immunoprecipitates were analyzed for enzymatic activity (see Fig. 2A) or phosphotyrosine (Top). Lanes 1, 2, and 5 contain ITK without c-Src; lanes 3, 4, and 6 contain ITK with c-Src. Lane 6 contains ITK cotransfected with the kinase-inactive c-Src. Lanes 2 and 4 were from cells incubated with Ly294002. (D) Inhibition of Src-induced tyrosine phosphorylation of ITK by wortmannin. COS-7 cells transfected with untagged wild-type ITK in the presence or absence of wild-type c-Src as indicated. ITK was immunoprecipitated and analyzed for phosphotyrosine (Top). Lanes: 1 and 3, ITK without c-Src; 2 and 4, ITK with c-Src. Lanes 3 and 6 were from cells incubated with wortmannin. Probes: (C and D Top) anti-phosphotyrosine antibodies; (Middle) anti-ITK antibodies; and (Bottom) anti-Src antibodies on whole cell lysates. Arrow indicates ITK or c-Src.
Figure 2
Figure 2
Enzymatic activity of ITK and mutants in the presence or absence of Src and inhibitor. (A) Immunoprecipitates of ITK or mutants expressed in the presence of absence or Src and/or Ly294002 were assayed for kinase activity. ▪, ITK alone; ░⃞, ITK plus Src; □, ITK plus Ly294002. Assays were corrected for expression and expressed as n-fold increase over ITK expressed in the absence of Src. (B) Enzymatic activity of ITK and mutants in the presence or absence of Ly294002. COS-7 cells transfected with the indicated mutants and either left untreated or treated with Ly294002. ITK immunoprecipitates were assayed for in vitro kinase activity, then probed with anti-HA antibody (to detect ITK) (Lower). The filter was treated with KOH and exposed to x-ray film (Upper). Arrow indicates ITK or mutants.
Figure 3
Figure 3
Overexpression of the p85 subunit of PI 3-kinase inhibits Src-induced tyrosine phosphorylation of ITK. ITK immunoprecipitates from COS-7 cells transfected with HA-tagged wild-type ITK in the presence or absence of c-Src and the p85 subunit of PI 3-kinase as indicated, were analyzed for phosphotyrosine (Top). Lanes: 1, ITK without c-Src; 2 and 3, ITK with c-Src; 3, ITK with c-Src and p85. (Upper Middle) Anti-HA immunoblot of the first panel demonstrating expression of ITK. (Lower Middle) Whole cell lysates probed for Src. (Bottom) Whole cell lysates probed for p85. Arrows indicate ITK, Src, and p85, respectively.
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.
Figure 5
Figure 5
Proposed mechanism of activation of ITK by Src. See Materials and Methods for details. The arrow from Src to ITK is meant to indicate that Src acts on ITK in some fashion to induce its tyrosine phosphorylation and activation. Curved arrows are meant to indicate one enzyme acting on the other and straight arrows indicate the next step in the activation of ITK by Src.
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