Kinase-independent functions for Itk in TCR-induced regulation of Vav and the actin cytoskeleton.
The Tec family kinase Itk is an important regulator of Ca(2+) 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.
Dombroski
Derek
D
National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA.
Houghtling
Richard A
RA
Labno
Christine M
CM
Precht
Patricia
P
Takesono
Aya
A
Caplen
Natasha J
NJ
Billadeau
Daniel D
DD
Wange
Ronald L
RL
Burkhardt
Janis K
JK
Schwartzberg
Pamela L
PL
D000199:Actins / Q000235:genetics / Q000378:metabolism*; D000818:Animals; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D019169:Jurkat Cells; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D010766:Phosphorylation; D025941:Protein Interaction Mapping; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D051567:Proto-Oncogene Proteins c-vav; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism; D011948:Receptors, Antigen, T-Cell / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics / Q000276:immunology; D013601:T-Lymphocytes / Q000201:enzymology / Q000378:metabolism; D014162:Transfection
D000199:Actins; D018797:Cell Cycle Proteins; D008565:Membrane Proteins; D011518:Proto-Oncogene Proteins; D051567:Proto-Oncogene Proteins c-vav; D034741:RNA, Small Interfering; D011948:Receptors, Antigen, T-Cell; C496618:VAV1 protein, human; C496619:Vav1 protein, mouse; D011505:Protein-Tyrosine Kinases; C080813:emt protein-tyrosine kinase