Itk Functions to Control Actin Polymerization at the Immune Synapse through Localized Activation of Cdc42 and WASP

Labno, Christine M.; Lewis, Carol M.; You, Daoqi; Leung, Daisy W.; Takesono, Ana; Kamberos, Natalie L.; Seth, Abhinav; Finkelstein, Lisa D.; Rosen, Michael K.; Schwartzberg, Pamela L.; Burkhardt, Janis K.

doi:10.1016/j.cub.2003.08.005

Cited by 124 publications

(116 citation statements)

References 31 publications

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“…We have previously reported that Itk-deficient cells show defective TCR-induced actin polarization that is associated with decreased activation of Cdc42, a key regulator of the WiskottAldrich syndrome protein (19). We additionally found that Vav was not properly targeted to the site of TCR contact.…”

Section: Discussionmentioning

confidence: 86%

“…T cell/bead conjugates were stained with Alexa594-phalloidin (Molecular Bioprobes) as described previously (19). For Vav localization, cells were stained with anti-FLAG (1:500 in 1.25% BSA/0.1% saponin in PBS at room temperature for 40 min), followed by two washes and incubation with a solution of FITC-anti-mouse IgG (1:1000) and Alexa594-phalloidin (1: 67) (19).…”

Section: Immunofluorescence Staining and Microscopymentioning

confidence: 99%

“…For Vav localization, cells were stained with anti-FLAG (1:500 in 1.25% BSA/0.1% saponin in PBS at room temperature for 40 min), followed by two washes and incubation with a solution of FITC-anti-mouse IgG (1:1000) and Alexa594-phalloidin (1: 67) (19). Conjugates were examined on an inverted Zeiss Axiophot microscope with ϫ40 and ϫ100 oil immersion objectives.…”

Section: Immunofluorescence Staining and Microscopymentioning

confidence: 99%

“…We have recently found that the altered actin polarization in Itk Ϫ/Ϫ T cells correlates with defective Cdc42 activation and a failure to localize Vav to the site of TCR stimulation, despite relatively normal TCRinduced Vav phosphorylation (19,21). To determine whether Vav localization correlated with the ability of Itk mutants to rescue actin polarization, we expressed a FLAG-tagged version of Vav, which facilitated subcellular localization studies.…”

Section: Regulation Of Vav Localizationmentioning

confidence: 99%

“…Notably, Itk is required for generating appropriate Th2 responses in vivo, a defect that may be linked to impaired activation of the Ca 2ϩ -sensitive NFAT transcription factors and/or altered T-bet expression (14 -17). More recently, Itk-deficient cells have been shown to have defects in actin polarization and the localized activation of the Rho family GTPase Cdc42 (18,19). Overexpression of the Itk pleckstrin homology (PH) domain or Itk containing a mutation affecting the Src homology 2 (SH2) protein interaction domain in Jurkat cells also prevents TCR-induced actin polymerization (18,20), suggesting that the Tec kinases may have important functions in the regulation of the actin cytoskeleton.…”

mentioning

confidence: 99%

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Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton

Dombroski

Houghtling

Labno

et al. 2005

The Journal of Immunology

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The Tec family kinase Itk is an important regulator of Ca2+ mobilization and is required for in vivo responses to Th2-inducing agents. Recent data also implicate Itk in TCR-induced regulation of the actin cytoskeleton. We have evaluated the requirements for Itk function in TCR-induced actin polarization. Reduction of Itk expression via small interfering RNA treatment of the Jurkat human T lymphoma cell line or human peripheral blood T cells disrupted TCR-induced actin polarization, a defect that correlated with decreased recruitment of the Vav guanine nucleotide exchange factor to the site of Ag contact. Vav localization and actin polarization could be rescued by re-expression of either wild-type or kinase-inactive murine Itk but not by Itk containing mutations affecting the pleckstrin homology or Src homology 2 domains. Additionally, we find that Itk is constitutively associated with Vav. Loss of Itk expression did not alter gross patterns of Vav tyrosine phosphorylation but appeared to disrupt the interactions of Vav with SLP-76. Expression of membrane-targeted Vav, Vav-CAAX, can rescue the small interfering RNA to Itk-induced phenotype, implicating the alteration in Vav localization as directly contributing to the actin polarization defect. These data suggest a kinase-independent scaffolding function for Itk in the regulation of Vav localization and TCR-induced actin polarization.

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Section: Discussionmentioning

confidence: 86%

Section: Immunofluorescence Staining and Microscopymentioning

confidence: 99%

Section: Immunofluorescence Staining and Microscopymentioning

confidence: 99%

Section: Regulation Of Vav Localizationmentioning

confidence: 99%

mentioning

confidence: 99%

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Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton

Dombroski

Houghtling

Labno

et al. 2005

The Journal of Immunology

Self Cite

124

132

View full text Add to dashboard Cite

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Impaired T‐cell development in the absence of Vav1 and Itk

et al. 2008

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Vav1 and the Tec family kinase Itk act in similar T‐cell activation pathways. Both molecules interact with members of the Cbl family of E3 ubiquitin ligases, and signaling defects in Vav1−/− T cells are rescued upon deletion of Cbl‐b. In this study we investigate the relation between Itk and Cbl‐b or Vav1 by generating Itk/Cbl‐b and Itk/Vav1 double‐deficient mice. Deletion of Cbl‐b in Itk−/− CD4+ T cells restored proliferation and partially IL‐2 production, and also led to a variable rescue of IL‐4 production. Thus, Itk and Vav1 act mechanistically similarly in peripheral T cells, since the defects in Itk−/− T cells, as in Vav1−/− T cells, are rescued if cells are released from the negative regulation mediated by Cbl‐b. In addition, only few peripheral CD4+ and CD8+ T cells were present in Vav1−/−Itk−/− mice due to severely impaired thymocyte differentiation. Vav1−/−Itk−/− thymocyte numbers were strongly reduced compared with WT, Itk−/− or Vav1−/− mice, and double‐positive thymocytes displayed increased cell death and impaired positive selection. Therefore, our data also reveal that the combined activity of Vav1 and Itk is required for proper T‐cell development and the generation of the peripheral T‐cell pool.

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Global analysis of dynamic changes in lipid raft proteins during T‐cell activation

et al. 2007

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Lipid rafts are considered as specialized microdomains within the plasma membrane with unique lipid compositions different from surrounding membranes. Following T-cell receptor (TCR) stimulation, lipid rafts assemble in T-cell/antigen-presenting cell (APC) contact site known as the immunological synapse, inner leaflets of which serve as activation or docking sites for downstream signaling components. To understand the signaling events occurring in lipid rafts, we globally analyzed dynamic changes in lipid raft proteins during TCR/CD28 costimulation using 2-D fluorescence difference gel electrophoresis. We detected multiple spots whose intensities were enhanced after costimulation, and identified proteins in these spots by PMF. Identified proteins include Src family tyrosine kinases, tyrosine phosphatase, phosphatidylinositol 3-kinase (PI3-kinase), actin-binding proteins, and regulators for small GTPases. Of particular interest, a number of pleckstrin homology (PH) domain-containing proteins were identified. Biochemical and histochemical analyses confirmed the translocation of these proteins from cytosol to lipid rafts. We also demonstrated that these proteins assembled at the T-cell/APC interface. These results indicate the efficacy of our system to systematically analyze dynamics of lipid raft proteins during extracellular stimulation.

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Itk Functions to Control Actin Polymerization at the Immune Synapse through Localized Activation of Cdc42 and WASP

Cited by 124 publications

References 31 publications

Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton

Kinase-Independent Functions for Itk in TCR-Induced Regulation of Vav and the Actin Cytoskeleton

Impaired T‐cell development in the absence of Vav1 and Itk

Global analysis of dynamic changes in lipid raft proteins during T‐cell activation

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