Targeting Pyk2 to β1-Integrin–Containing Focal Contacts Rescues Fibronectin-Stimulated Signaling and Haptotactic Motility Defects of Focal Adhesion Kinase–Null Cells

Abstract

Focal adhesion kinase–null (FAK−/−) fibroblasts exhibit morphological and motility defects that are reversed by focal adhesion kinase (FAK) reexpression. The FAK-related kinase, proline-rich tyrosine kinase 2 (Pyk2), is expressed in FAK−/− cells, yet it exhibits a perinuclear distribution and does not functionally substitute for FAK. Chimeric Pyk2/FAK proteins were created and expressed in FAK−/− cells to determine the impact of Pyk2 localization to focal contacts. Whereas an FAK/Pyk2 COOH-terminal (CT) domain chimera was perinuclear distributed, stable expression of a Pyk2 chimera with the FAK-CT domain (Pyk2/FAK-CT) localized to focal contact sites and enhanced fibronectin (FN)-stimulated haptotactic cell migration equal to FAK-reconstituted cells. Disruption of paxillin binding to the FAK-CT domain (S-1034) inhibited Pyk2/FAK-CT localization to focal contacts and its capacity to promote cell motility. Paxillin binding to the FAK-CT was necessary but not sufficient to mediate the indirect association of FAK or Pyk2/FAK-CT with a β1-integrin–containing complex. Both FAK and Pyk2/FAK-CT but not Pyk2/FAK-CT S-1034 reconstituted FAK−/− cells, exhibit elevated FN-stimulated extracellular signal–regulated kinase 2 (ERK2) and c-Jun NH2-terminal kinase (JNK) kinase activation. FN-stimulated FAK or Pyk2/FAK-CT activation enhanced both the extent and duration of FN-stimulated ERK2 activity which was necessary for cell motility. Transient overexpression of the FAK-CT but not FAK-CT S-1034 domain inhibited both FN-stimulated ERK2 and JNK activation as well as FN-stimulated motility of Pyk2/FAK-CT reconstituted cells. These gain-of-function studies show that the NH2-terminal and kinase domains of Pyk2 can functionally substitute for FAK in promoting FN-stimulated signaling and motility events when localized to β-integrin–containing focal contact sites via interactions mediated by the FAK-CT domain.

Soluble Fractalkine Prevents Monocyte Chemoattractant Protein-1-Induced Monocyte Migration via Inhibition of Stress-Activated Protein Kinase 2/p38 and Matrix Metalloproteinase Activities

“…It is now well established that the focal adhesion kinase (FAK) tyrosine kinase family plays a crucial role in the signaling pathways implicated during the cellular adherence and migration (42)(43)(44). We first observed that, as demonstrated in freshly isolated human monocytes (45,46) and in THP1 cells (14), the human monocytic MonoMac cells express only the related FAK tyrosine kinase Pyk2 and not p125 FAK (Fig.…”

“…Members of the FAK family have been shown to play a key role in the control of the adhesion/migration process (42)(43)(44) and also to intervene in the control of MAPK pathways (47, 49 -51). We found that FAK is not expressed in MonoMac6 cells, whereas Pyk2, which is highly expressed in this cell line, was rapidly stimulated in response to MCP-1, in accord with previous data obtained on THP-1 cells (14) and on freshly isolated monocytes with RANTES (45).…”

Nuclear FAK: a New Mode of Gene Regulation from Cellular Adhesions

“…Pyk2, another member of FAK family kinase, is highly expressed in hematopoietic, endothelial, and neuronal cells (Dikic and Schlessinger, 1998;Lev et al, 1995). Although both kinases exhibit structural similarities, their subcellular localization and functions are distinct (Klingbeil et al, 2001).…”

“…1). FAK C-terminal domain is called as focal adhesion targeting (FAT) domain which contains binding sites for both paxillin and talin, thereby connects to cellular adhesion sites called focal adhesions (FAs) while the C-terminal domain of Pyk2 does not have talin binding motif and do not normally localize to the FAs (Klingbeil et al, 2001;Lawson et al, 2012). This difference makes Pyk2 subcellular localization more favorable to perinuclear region than FAs.…”

Optimal Intensity Shock Wave Promotes the Adhesion and Migration of Rat Osteoblasts via Integrin β1-mediated Expression of Phosphorylated Focal Adhesion Kinase

“…The beneficial effects for enhanced osteoblast adhesion and migration may result from reorganization of actin cytoskeleton to a certain degree (23). The regulation of cell function by actin cytoskeleton is complex and may also involve integrin-mediated matrix binding and signal pathways (62)(63)(64)(65)(66). In addition, cells need to spread fully during migration (45), and the enhanced ability of osteoblasts to spread would promote cell migration.…”