Submitted on January 24, 2008
Revised on March 25, 2008
Pyk2 Regulates Spreading and Migration of Eosinophils after 
2 Integrin Adhesion
Xiangdong Zhu1*, Evan Boetticher1, Lin Wang1, Yingli Duan1, Jonathan Learoyd1, and Alan R Leff2
1 Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, IL, USA,
2 Department of Medicine, Section of Pulmonary and Critical Care Medicine, University of Chicago, Chicago, IL, USA; Department of Neurobilogy, Pharmacology and Physiology, Pediatrics, Anesthesia and Critical Care, and Committees on Clinical Pharmacology, Cell Physiology and Molecular Medicine, Division of Biological Sciences, University of Chicago, Chicago, IL, USA
* To whom correspondence should be addressed. E-mail: XZhu{at}medicine.bsd.uchicago.edu.
We examined the role of Pyk2 in spreading and migration of human blood eosinophils after 
2-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, PAF, fMLP or Mn2+. To determine the role of Pyk2 in regulating eosinophil migration, we utilized a transducable dominant-negative inhibitor of 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 2-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, while non-spreading 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, while not involved in 2-integrin adhesion, causes eosinophil spreading and regulates subsequent chemotactic migration after 2-integrin ligation to endothelial counter-ligands. We conclude that Pyk2 is activated by 2-integrin adhesion and a required signal for eosinophil spreading and subsequent chemotactic migration.
