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(Journal of Leukocyte Biology. 2002;71:520-530.)
© 2002 by Society for Leukocyte Biology

LFA-1 integrin and the microtubular cytoskeleton are involved in the Ca²⁺-mediated regulation of the activity of the tyrosine kinase PYK2 in T cells

José Luis Rodríguez-Fernández^*, Lorena Sánchez-Martín^*, Cristina Alvarez de Frutos^*, David Sancho, Martyn Robinson, Francisco Sánchez-Madrid and Carlos Cabañas^*

^* Instituto de Farmacología y Toxicología (Centro Mixto CSIC-UCM), Facultad de Medicina, Universidad Complutense, Madrid, Spain;
Servicio de Inmunología, Hospital de la Princesa, Madrid, Spain; and
Celltech Ltd., Slough, United Kingdom

Correspondence: Dr. Carlos Cabañas, Instituto de Farmacología y Toxicología (CSIC-UCM), Facultad de Medicina UCM, Pabellón III, 28040 Madrid, Spain. E-mail: cacabagu{at}med.ucm.es

Lymphocyte function-associated antigen (LFA-1) is a member of the ß2 family of integrins that is selectively expressed on leukocytes. Herein, we show that Ca²⁺ 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, Ca²⁺ mobilization did not induce activation of PYK2 when the LFA-1/ICAM-1 interaction was prevented with function-blocking mAb, implying that the Ca²⁺-induced activation of PYK2 requires integrin engagement. Furthermore, pretreatment of the cells with the Ca²⁺ chelator EGTA, which depletes the intracellular Ca²⁺, 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 Ca²⁺ exerts its effects through a target downstream of this integrin. In this regard, immunofluorescence and Western blot analysis showed that Ca²⁺ 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 Ca²⁺-mediated activation of PYK2 in T-lymphocytes.

Key Words: adhesion molecules • T-lymphocytes • protein kinases • signal transduction

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