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

Abstract

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.

Pyk2 Controls Integrin-Dependent CTL Migration through Regulation of De-Adhesion

“…In contrast, Pyk2 positively regulates naive mouse CD8 T cell (17) and NK cell (20) adhesion to ICAM-1, and has no impact on neutrophil (16) or eosinophil (21) adhesion. Knockout or inhibition of Pyk2 enhances macrophage cell spreading (15); decreases cell spreading of naive T cells (17), B cells (22), eosinophils (21), and HL-60 cells (23); and has no effect on neutrophil spreading (16).…”

Inhibition of Pyk2 Blocks Airway Inflammation and Hyperresponsiveness in a Mouse Model of Asthma.

“…Pyk2 regulates the formation of lamellapodia and filapodia as well as chemotaxis to IL-5, PAF, and fMLP (18). The objective of this investigation was to determine if these prior preliminary observations would translate to reduction in AHR in an immune-sensitized mouse in vivo.…”

“…Moreover, overexpression of the kinase-dead mutant of Pyk2 known to prevent Pyk2 enzymatic activity blocked IL-8-induced chemotaxis of HL-60-derived PMN-like cells (22). We have shown that while Pyk2 does not initiate endothelial adhesion of eosinophils, it is essential for eosinophil spreading and migration to various chemoattractants after b2 integrin adhesion in vitro (18). However, the role of Pyk2 in asthma and allergic inflammation in vivo is unknown.…”

“…We have found previously that Pyk2 is not involved in integrinmediated eosinophil adhesion, the first stage of cell migration. However, b2 integrin adhesion of eosinophils up-regulates Pyk2 activity, and Pyk2 inhibition blocks eosinophil chemotaxis to IL-5 or fMLP in vitro (18). The objective of this investigation was to determine the role of Pyk2 in eosinophil migration in immune-sensitized mice in vivo.…”

“…BL21(DE3)pLysS-competent Escherichia coli was transformed with either pTAT-Pyk2-CT or pTAT-GFP construct, and fusion proteins were produced using a native isolation method (18,27). Briefly, E. coli cells were sonicated in Tris-buffered saline plus protease inhibitor cocktail (Roche, Indianapolis, IN) and centrifuged, and the resulting supernatant was loaded onto a NiProBond resin column (Invitrogen, Carlsbad, CA).…”