Proline-rich tyrosine kinase 2 regulates spreading and migration of eosinophils after beta2-integrin adhesion.
We examined the role of proline-rich tyrosine kinase (Pyk) 2 in the spreading and migration of human blood eosinophils after beta(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, platelet-activating factor (PAF), formyl-met-leu-phe (fMLP), or Mn(2)(+). 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 beta(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, 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 beta(2)-integrin adhesion, causes eosinophil spreading and regulates subsequent chemotactic migration after beta(2)-integrin ligation to endothelial counter ligands. We conclude that Pyk2 is activated by beta(2)-integrin adhesion and is a required signal for eosinophil spreading and subsequent chemotactic migration.
Zhu
Xiangdong
X
Section of Pulmonary and Critical Care Medicine, Department of Medicine, MC6076, University of Chicago, 5841 South Maryland Avenue, Chicago, IL 60637, USA. xzhu@medicine.bsd.uchicago.edu
Boetticher
Evan
E
Wang
Lin
L
Duan
Yingli
Y
Learoyd
Jonathan
J
Leff
Alan R
AR
D015153:Blotting, Western; D018821:CD18 Antigens / Q000502:physiology*; D002448:Cell Adhesion / Q000502:physiology*; D002465:Cell Movement / Q000502:physiology*; D004804:Eosinophils / Q000166:cytology; D051418:Focal Adhesion Kinase 2 / Q000378:metabolism*; D006801:Humans; D010766:Phosphorylation; D014443:Tyrosine / Q000378:metabolism
D018821:CD18 Antigens; D014443:Tyrosine; D051418:Focal Adhesion Kinase 2