LFA-1 integrin and the microtubular cytoskeleton are involved in the Ca(2)(+)-mediated regulation of the activity of the tyrosine kinase PYK2 in T cells.
Lymphocyte function-associated antigen (LFA-1) is a member of the beta2 family of integrins that is selectively expressed on leukocytes. Herein, we show that Ca(2)(+) mobilizing agents A23187, thapsigargin, and ionomycin induce an increase in adhesion to the LFA-1 ligand intercellular adhesion molecule-1 (ICAM-1) and activation and redistribution of the proline-rich tyrosine kinase-2 (PYK2) to the microtubule-organizing center (MTOC) in T-lymphoblasts. These effects are similar to those observed upon direct induction of activation of LFA-1 with the stimulatory mAb KIM-127. Most importantly, Ca(2)(+) mobilization did not induce activation of PYK2 when the LFA-1/ICAM-1 interaction was prevented with function-blocking mAb, implying that the Ca(2)(+)-induced activation of PYK2 requires integrin engagement. Furthermore, pretreatment of the cells with the Ca(2)(+) chelator EGTA, which depletes the intracellular Ca(2)(+), inhibited the effects of mAb KIM-127 on cell morphology and PYK2 activation. This inhibition with EGTA was not reversed by cross-linking integrin LFA-1 with specific antibodies, indicating that Ca(2)(+) exerts its effects through a target downstream of this integrin. In this regard, immunofluorescence and Western blot analysis showed that Ca(2)(+) chelators affect the organization of the microtubular cytoskeleton and the localization of PYK2 to the MTOC area, suggesting that these agents could inhibit the activation of PYK2 by interfering with the microtubular network of T cells. Taken together, our results demonstrate for the first time an important role for the integrin LFA-1 and the microtubular cytoskeleton in the Ca(2)(+)-mediated activation of PYK2 in T-lymphocytes.
Rodríguez-Fernández
José Luis
JL
Instituto de Farmacología y Toxicología (Centro Mixto CSIC-UCM), Facultad de Medicina, Universidad Complutense, Pabellón III, 28040 Madrid, Spain.
Sánchez-Martín
Lorena
L
de Frutos
Cristina Alvarez
CA
Sancho
David
D
Robinson
Martyn
M
Sánchez-Madrid
Francisco
F
Cabañas
Carlos
C
D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D002478:Cells, Cultured; D003599:Cytoskeleton / Q000502:physiology* / Q000648:ultrastructure; D051418:Focal Adhesion Kinase 2; D006801:Humans; D018799:Intercellular Adhesion Molecule-1 / Q000502:physiology; D016169:Lymphocyte Function-Associated Antigen-1 / Q000502:physiology*; D008870:Microtubules / Q000502:physiology / Q000648:ultrastructure; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D015398:Signal Transduction / Q000502:physiology; D013601:T-Lymphocytes / Q000378:metabolism* / Q000648:ultrastructure
D016169:Lymphocyte Function-Associated Antigen-1; D018799:Intercellular Adhesion Molecule-1; D011505:Protein-Tyrosine Kinases; D051418:Focal Adhesion Kinase 2; D002118:Calcium