ITK-SH3-mediated interactions, both with exogenous ligands and via intermolecular self-association with ITK-SH2, have been shown to be important for regulation of ITK activity. The biological significance of these competing SH3 interactions is not completely understood. A mutant of ITK where substitution of the SH3 domain with that of the related kinase BTK (ITK-BTK(SH3)) was used to disrupt intermolecular self-association of ITK while maintaining canonical binding to exogenous ligands such as SLP-76. ITK-BTK(SH3) displays reduced association with SLP-76 leading to inefficient transphosphorylation, reduced phosphorylation of PLCγ1, and diminished Th2 cytokine production. In contrast, ITK-BTK(SH3) displays no defect in its localization to the T-cell-APC contact site. Another mutation, Y511F, in the activation loop of ITK, impairs ITK activation. T cells expressing ITK-Y511F display defective phosphorylation of ITK and its downstream target PLCγ1, as well as significant inhibition of Th2 cytokines. In contrast, the inducible localization of ITK-Y511F to the T cell-APC contact site and its association with SLP-76 are not affected. The presented data lend further support to the hypothesis that precise interactions between ITK and its signaling partners are required to support ITK signaling downstream of the TCR. 1. Introduction Protein tyrosine kinases play a critical role in signaling through the T-cell Antigen Receptor/CD3 (TCR-CD3) molecular complex [1]. The Tec family of tyrosine kinases regulates lymphocyte development, differentiation, and activation [2]. Among them, the Inducible T-Cell Kinase (ITK) plays an important role in T-cell signaling and function [3]. ITK is the major Tec kinase expressed in T cells, regulating intracellular Ca++ mobilization by phosphorylation and activation of Phospholipase Cγ1 [4], TCR-induced actin polymerization [5], and production of Th2 cytokines [6]. Even though ITK does not appear to be important for the development of？？Th2 cells per se, it is critical for the expression of Th2 cell effector function, as evidenced by defective expression of Th2 cytokines in ITK-deficient animals [7]. ITK is structurally organized into domains that carry out its catalytic function, domains that regulate its catalytic activity, and domains that provide sites for interactions with other signaling partners [8]. Even though the crystal structure of full-length ITK has not been resolved, important insights into its structure have been provided by NMR-based analysis of individual ITK domains [2]. Previous in vitro observations provide some
References
[1]
C. S Guy and D. A. A Vignali, “Organization of proximal signal initiation at the TCR:CD3 complex,” Immunological Reviews, vol. 232, no. 1, pp. 7–21, 2009.
[2]
A. H Andreotti, P. L Schwartzberg, R. E Joseph, and L. J Berg, “T-cell signaling regulated by the Tec family kinase, Itk,” Cold Spring Harbor perspectives in biology, vol. 2, no. 7, article a002287, 2010.
[3]
L. J Berg, L. D Finkelstein, J. A Lucas, and P. L Schwartzberg, “Tec family kinases in T lymphocyte development and function,” Annual Review of Immunology, vol. 23, pp. 549–600, 2005.
[4]
D Beach, R Gonen, Y Bogin, I. G Reischl, and D Yablonski, “Dual role of SLP-76 in mediating T cell receptor-induced activation of phospholipase C-γ1,” Journal of Biological Chemistry, vol. 282, no. 5, pp. 2937–2946, 2007.
[5]
C. D Tsoukas, J. A Grasis, C. D Browne, and K. A Ching, “Inducible T cell tyrosine kinase (ITK): structural requirements and actin polymerization,” Advances in Experimental Medicine and Biology, vol. 584, pp. 29–41, 2006.
[6]
J. A Grasis and C. D Tsoukas, “Itk: the rheostat of the T cell response,” Journal of Signal Transduction, vol. 2011, Article ID 297868, 23 pages, 2011.
[7]
B. B Au-Yeung, S. D Katzman, and D. J Fowell, “Cutting edge: Itk-dependent signals required for CD4+ T cells to exert, but not gain, Th2 effector function,” Journal of Immunology, vol. 176, no. 7, pp. 3895–3899, 2006.
[8]
W. C Yang, Y Collette, J. A Nunès, and D Olive, “Tec kinases: a family with multiple roles in immunity,” Immunity, vol. 12, no. 4, pp. 373–382, 2000.
[9]
S. C Bunnell, M Diehn, M. B Yaffe, P. R Findell, L. C Cantley, and L. J Berg, “Biochemical interactions integrating Itk with the T cell receptor-initiated signaling cascade,” Journal of Biological Chemistry, vol. 275, no. 3, pp. 2219–2230, 2000.
[10]
M. S Jordan, J. E Smith, J. C Burns et al., “Complementation in trans of altered thymocyte development in mice expressing mutant forms of the adaptor molecule SLP76,” Immunity, vol. 28, no. 3, pp. 359–369, 2008.
[11]
J. A Grasis, D. M Guimond, N. R Cam et al., “In vivo significance of ITK-SLP-76 interaction in cytokine production,” Molecular and Cellular Biology, vol. 30, no. 14, pp. 3596–3609, 2010.
[12]
K. N Brazin, D. B Fulton, and A. H Andreotti, “A specific intermolecular association between the regulatory domains of a Tec family kinase,” Journal of Molecular Biology, vol. 302, no. 3, pp. 607–623, 2000.
[13]
L Min, W Wu, R. E Joseph, D. B Fulton, L Berg, and A. H Andreotti, “Disrupting the intermolecular self-association of Itk enhances T cell signaling,” Journal of Immunology, vol. 184, no. 8, pp. 4228–4235, 2010.
[14]
K. A Ching, Y Kawakami, T Kawakami, and C. D Tsoukas, “Emt/Itk associates with activated TCR complexes: role of the pleckstrin homology domain,” Journal of Immunology, vol. 163, no. 11, pp. 6006–6013, 1999.
[15]
S. D Heyeck, H. M Wilcox, S. C Bunnell, and L. J Berg, “Lck phosphorylates the activation loop tyrosine of the Itk kinase domain and activates Itk kinase activity,” Journal of Biological Chemistry, vol. 272, no. 40, pp. 25401–25408, 1997.
[16]
T Zal and N. R. J Gascoigne, “Photobleaching-corrected FRET efficiency imaging of live cells,” Biophysical Journal, vol. 86, no. 6, pp. 3923–3939, 2004.
[17]
M. C Montoya, D Sancho, G Bonello et al., “Role of ICAM-3 in the initial interaction of T lymphocytes and APCs,” Nature Immunology, vol. 3, no. 2, pp. 159–168, 2002.
[18]
K. R Schulz, E. A Danna, P. O Krutzik, and G. P Nolan, “Single-cell phospho-protein analysis by flow cytometry,” Current Protocols in Immunology, vol. 8, unit 8.17, 2007.
[19]
P. O Krutzik and G. P Nolan, “Intracellular phospho-protein staining techniques for flow cytometry: monitoring single cell signaling events,” Cytometry Part A, vol. 55, no. 2, pp. 61–70, 2003.
[20]
I. C Ho, D Lo, and L. H Glimcher, “c-maf Promotes T helper cell type 2 (Th2) and attenuates Th1 differentiation by both interleukin 4-dependent and -independent mechanisms,” Journal of Experimental Medicine, vol. 188, no. 10, pp. 1859–1866, 1998.
[21]
A. T Miller, H. M Wilcox, Z Lai, and L. J Berg, “Signaling through Itk promotes T helper 2 differentiation via negative regulation of T-bet,” Immunity, vol. 21, no. 1, pp. 67–80, 2004.
[22]
H. M Wilcox and L. J Berg, “Itk phosphorylation sites are required for functional activity in primary T cells,” Journal of Biological Chemistry, vol. 278, no. 39, pp. 37112–37121, 2003.
[23]
M. I Wahl, A. C Fluckiger, R. M Kato, H Park, O. N Witte, and D. J Rawlings, “Phosphorylation of two regulatory tyrosine residues in the activation of Bruton's tyrosine kinase via alternative receptors,” Proceedings of the National Academy of Sciences of the United States of America, vol. 94, no. 21, pp. 11526–11533, 1997.
[24]
B Poulin, F Sekiya, and S. G Rhee, “Intramolecular interaction between phosphorylated tyrosine-783 and the C-terminal Src homology 2 domain activates phospholipase C-γ1,” Proceedings of the National Academy of Sciences of the United States of America, vol. 102, no. 12, pp. 4276–4281, 2005.
[25]
A Baguet and M Bix, “Chromatin landscape dynamics of the Il4-Il13 locus during T helper 1 and 2 development,” Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 31, pp. 11410–11415, 2004.
[26]
Y. H Huang, J. A Grasis, A. T Miller et al., “Positive regulation of Itk PH domain function by soluble IP4,” Science, vol. 316, no. 5826, pp. 886–889, 2007.
[27]
K. A Ching, J. A Grasis, P Tailor, Y Kawakami, T Kawakami, and C. D Tsoukas, “TCR/CD3-induced activation and binding of Emt/Itk to linker of activated T cell complexes: requirement for the Src homology 2 domain,” Journal of Immunology, vol. 165, no. 1, pp. 256–262, 2000.
[28]
D Dombroski, R. A Houghtling, C. M Labno et al., “Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton,” Journal of Immunology, vol. 174, no. 3, pp. 1385–1392, 2005.
[29]
Q Qi, N Sahu, and A August, “Tec kinase Itk forms membrane clusters specifically in the vicinity of recruiting receptors,” Journal of Biological Chemistry, vol. 281, no. 50, pp. 38529–38534, 2006.
