Alternative splicing controls the mechanisms of FAK autophosphorylation.
Focal adhesion kinase (FAK) is activated following integrin engagement or stimulation of transmembrane receptors. Autophosphorylation of FAK on Tyr-397 is a critical event, allowing binding of Src family kinases and activation of signal transduction pathways. Tissue-specific alternative splicing generates several isoforms of FAK with different autophosphorylation rates. Despite its importance, the mechanisms of FAK autophosphorylation and the basis for differences between isoforms are not known. We addressed these questions using isoforms of FAK expressed in brain. Autophosphorylation of FAK(+), which is identical to that of "standard" FAK, was intermolecular in transfected cells, although it did not involve the formation of stable multimeric complexes. Coumermycin-induced dimerization of gyrase B-FAK(+) chimeras triggered autophosphorylation of Tyr-397. This was independent of cell adhesion but required the C terminus of the protein. In contrast, the elevated autophosphorylation of FAK(+6,7), the major neuronal splice isoform, was not accounted for by transphosphorylation. Specifically designed immune precipitate kinase assays confirmed that autophosphorylation of FAK(+) was intermolecular, whereas autophosphorylation of FAK(+6,7) or FAK(+7) was predominantly intramolecular and insensitive to the inhibitory effects of the N-terminal domain. Our results clarify the mechanisms of FAK activation and show how alternative splicing can dramatically alter the mechanism of autophosphorylation of a protein kinase.
Toutant
Madeleine
M
INSERM/UPMC U536, Institut du Fer à Moulin, 75005 Paris, France.
Costa
Alicia
A
Studler
Jeanne-Marie
JM
Kadaré
Gress
G
Carnaud
Michèle
M
Girault
Jean-Antoine
JA
D017398:Alternative Splicing; D049933:Aminocoumarins; D000818:Animals; D001921:Brain / Q000201:enzymology; D019556:COS Cells; D003374:Coumarins / Q000378:metabolism; D027081:DNA Gyrase / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D004791:Enzyme Inhibitors / Q000378:metabolism; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008954:Models, Biological; D016297:Mutagenesis, Site-Directed; D009675:Novobiocin / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000235:genetics / Q000378:metabolism*; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D059005:Topoisomerase II Inhibitors; D014443:Tyrosine / Q000378:metabolism
D049933:Aminocoumarins; D003374:Coumarins; D004791:Enzyme Inhibitors; D007527:Isoenzymes; D010455:Peptides; D059005:Topoisomerase II Inhibitors; D009675:Novobiocin; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D027081:DNA Gyrase; C004628:coumermycin