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© The Rockefeller University Press,
0021-9525/2001//97 $5.00
The Journal of Cell Biology, Volume 152, Number 1,
, 2001 97-110
Original Article |
Targeting Pyk2 to β1-Integrin–Containing Focal Contacts Rescues Fibronectin-Stimulated Signaling and Haptotactic Motility Defects of Focal Adhesion Kinase–Null Cells
dschlaep{at}scripps.edu
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.
Key Words: FAK • Pyk2 • cell migration • integrins • signaling
© 2001 The Rockefeller University Press
C.K. Klingbeil and C.R. Hauck contributed equally to this work.Abbreviations used in this paper: β-galactosidase, β-gal; CT, COOH-terminal; ERK, extracellular signal–regulated kinase; FAK, focal adhesion kinase; FAT, focal adhesion targeting; FERM, band 4.1, erzin, radixin, and moesin; FN, fibronectin; FRNK, FAK-related nonkinase; HA, hemagglutinin; IP, immunoprecipitation; JNK, c-Jun NT kinase; MBP, myelin basic protein; MEK, mitogen-activated protein kinase; NT, NH2-terminal; P.Tyr, phosphotyrosine; PTK, protein tyrosine kinase; Pyk2, proline-rich tyrosine kinase 2; SH, Src homology; WCL, whole cell lysate.
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