In situ photoactivation of a caged phosphotyrosine peptide derived from focal adhesion kinase temporarily halts lamellar extension of single migrating tumor cells.
Focal adhesion kinase (FAK), a non-receptor tyrosine kinase, mediates integrin-based cell signaling by transferring signals regulating cell migration, adhesion, and survival from the extracellular matrix to the cytoplasm. Following autophosphorylation at tyrosine 397, FAK binds the Src homology 2 domains of Src and phosphoinositide 3-kinase, among several other possible binding partners. To further investigate the role of phosphorylated FAK in cell migration in situ, peptides comprising residues 391-406 of human FAK with caged phosphotyrosine 397 were synthesized. Although the caged phosphopeptides were stable to phosphatase activity, the free phosphopeptides showed a half-life of approximately 10-15 min in cell lysates. Migrating NBT-II cells (a rat bladder tumor cell line) were microinjected with the caged FAK peptide and locally photoactivated using a focused laser beam. The photoactivation of caged FAK peptide in 8-microm diameter spots over the cell body led to the temporary arrest of the leading edge migration within approximately 1 min of irradiation. In contrast, cell body migration was not inhibited. Microinjection of a non-caged phosphorylated tyrosine 397 FAK peptide into migrating NBT-II cells also led to lamellar arrest; however, this approach lacks the temporal control afforded by the caged phosphopeptides. Photoactivation of related phosphotyrosine peptides with altered sequences did not result in transient lamellar arrest. We hypothesize that the phosphorylated FAK peptide competes with the endogenous FAK for binding to FAK effectors including, but not limited to, Src and phosphoinositide 3-kinase, causing spatiotemporal misregulation and subsequent lamellar arrest.
Humphrey
David
D
Department of Cell and Developmental Biology, Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
Rajfur
Zenon
Z
Vazquez
M Eugenio
ME
Scheswohl
Danielle
D
Schaller
Michael D
MD
Jacobson
Ken
K
Imperiali
Barbara
B
D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002448:Cell Adhesion; D045744:Cell Line, Tumor; D002465:Cell Movement; D002470:Cell Survival; D002851:Chromatography, High Pressure Liquid; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D008027:Light; D008956:Models, Chemical; D010455:Peptides / Q000737:chemistry; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000737:chemistry*; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D015398:Signal Transduction; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
D010455:Peptides; D011993:Recombinant Fusion Proteins; D019000:Phosphotyrosine; D014443:Tyrosine; D005982:Glutathione Transferase; D019869:Phosphatidylinositol 3-Kinases; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat