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Proline-Rich Tyrosine Kinase 2 Regulates Spreading and Migration of Eosinophils after β₂-Integrin Adhesion

¹ Section of Pulmonary and Critical Care Medicine, Department of Medicine, and ² Departments of Neurobiology, Pharmacology and Physiology, Pediatrics, Anesthesia, and Critical Care, and Committees on Clinical Pharmacology, Cell Physiology, and Molecular Medicine, Division of the Biological Sciences, University of Chicago, Chicago, Illinois

Correspondence and requests for reprints should be addressed to Xiangdong Zhu, M.D., Section of Pulmonary and Critical Care Medicine, Department of Medicine, MC6076, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637. E-mail: xzhu{at}medicine.bsd.uchicago.edu

We examined the role of proline-rich tyrosine kinase (Pyk) 2 in the spreading and migration of human blood eosinophils after β₂-integrin ligation. Western blot analysis showed that Pyk2 was activated by phosphorylation at Y402 after eosinophil adhesion to BSA-coated plates after activation with IL-5, platelet-activating factor (PAF), formyl-met-leu-phe (fMLP), or Mn²⁺. To determine the role of Pyk2 in regulating eosinophil migration, we used a transducable dominant-negative inhibitor of Pyk2, TAT-mediated protein transduction of dominant-negative C-terminal Pyk2 (TAT-Pyk2-CT), a fusion protein in which TAT peptide was fused to the C-terminal Pyk2. TAT-Pyk2-CT blocked tyrosine phosphorylation of Pyk2 caused by β₂-integrin adhesion, but did not block adhesion of eosinophils to plated BSA. TAT-Pyk2-CT also blocked subsequent spreading and migration of eosinophils caused by IL-5, PAF, or fMLP. Spreading eosinophils stained with FITC-conjugated phalloidin showed elongation and formation of multiple fillopodia and lamellipodia, whereas nonspreading eosinophils were smaller and round. Treatment of eosinophils with TAT-Pyk2-CT had no effect on the initial cell polarization, but blocked the formation of fillopodia and lamellipodia in adherent cells. Migration of eosinophils through Transwell plates caused by IL-5, PAF, or fMLP was blocked significantly after inhibition of Pyk2. These data indicate that Pyk2, although not involved in β₂-integrin adhesion, causes eosinophil spreading and regulates subsequent chemotactic migration after β₂-integrin ligation to endothelial counter ligands. We conclude that Pyk2 is activated by β₂-integrin adhesion and is a required signal for eosinophil spreading and subsequent chemotactic migration.

Key Words: eosinophils • chemotaxis • adhesion molecules • signal transduction

CLINICAL RELEVANCE This research focuses on the mechanisms by which the protein tyrosine kinase, proline-rich tyrosine kinase 2, regulates eosinophil spreading and chemotaxis. Data derived from this study should suggest novel approaches for antiinflammatory therapies in asthma and allergic diseases.