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Rho and Rho-associated kinase modulate the tyrosine kinase PYK2 in T-cells through regulation of the activity of the

J L Rodríguez-Fernández1, L Sánchez-Martín, M Rey

  • 1Instituto de Farmacología y Toxicología CSIC, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain.

The Journal of Biological Chemistry
|August 8, 2001
PubMed

Insights

The small GTPase Rho and Rho-associated kinase (ROCK) control T-cell adhesion and signaling by regulating lymphocyte function-associated antigen-1 (LFA-1) integrin activity. This regulation involves actin organization and PYK2 tyrosine kinase activation.

Area of Science:

  • Immunology
  • Cell Biology
  • Molecular Biology

Background:

  • The small GTPase Rho and its effector Rho-associated kinase (ROCK) are critical regulators of cellular processes.
  • Lymphocyte function-associated antigen-1 (LFA-1) is an integrin crucial for T-cell adhesion and immune responses.
  • The precise mechanisms by which Rho/ROCK influence LFA-1 function in T-cells remain incompletely understood.

Purpose of the Study:

  • To investigate the role of the small GTPase Rho and ROCK in regulating the adhesive and signaling functions of LFA-1 in human T-lymphocytes.
  • To elucidate the downstream signaling pathways affected by Rho/ROCK inhibition in the context of LFA-1 engagement.

Main Methods:

  • Human T-lymphocytes were treated with C3-exoenzyme or Y-27632 to inhibit Rho or ROCK, respectively.
  • Dominant-negative (N19Rho) or constitutively active (V14Rho) Rho mutants were introduced into T-cells via transfection.
  • Cellular adhesion assays, F-actin staining, and Western blotting for PYK2 activation were performed.
  • Experiments involved plating cells on ICAM-1, poly-l-lysine, or using anti-LFA-1 blocking antibodies.

Main Results:

  • Inhibition of Rho or ROCK led to F-actin disorganization and aggregation.
  • Rho/ROCK inhibition induced LFA-1-mediated adhesion to ICAM-1 via LFA-1 clustering, without altering expression or affinity.
  • T-cell polarization and PYK2 activation were induced upon Rho/ROCK inhibition specifically during LFA-1 engagement with ICAM-1.
  • Constitutively active Rho blocked LFA-1 clustering and PYK2 activation.

Conclusions:

  • The small GTPase Rho, through ROCK and F-actin, regulates LFA-1 integrin activity and subsequent PYK2 activation in T-cells.
  • This study presents a novel mechanism where Rho controls cytoplasmic tyrosine kinase activity via integrin regulation.
  • These findings offer new insights into the molecular control of T-cell adhesion and signaling.

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