Autophosphorylation of Tyr397 and its phosphorylation by Src-family kinases are altered in focal-adhesion-kinase neuronal isoforms.
In brain, focal adhesion kinase (FAK) is regulated by neurotransmitters and has a higher molecular mass than in other tissues, due to alternative splicing. Two exons code for additional peptides of six and seven residues ('boxes' 6 and 7), located on either side of Tyr(397), which increase its autophosphorylation. Using in situ hybridization and a monoclonal antibody (Mab77) which does not recognize FAK containing box 7, we show that, although mRNAs coding for boxes 6 and 7 have different patterns of expression in brain, FAK+6,7 is the main isoform in forebrain neurons. The various FAK isoforms fused to green fluorescent protein were all targeted to focal adhesions in non-neuronal cells. Phosphorylation-state-specific antibodies were used to study in detail the phosphorylation of Tyr(397), a critical residue for the activation and function of FAK. The presence of boxes 6 and 7 increased autophosphorylation of Tyr(397) independently and additively, whereas they had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-family kinases were also able to phosphorylate Tyr(397) in cells, but this phosphorylation was decreased in the presence of box 6 or 7, and abolished in the presence of both. Thus the additional exons characteristic of neuronal isoforms of FAK do not alter its targeting, but change dramatically the phosphorylation of Tyr(397). They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when co-transfected with Src-family kinases.
Toutant
M
M
INSERM U114, Collège de France, 11 place Marcelin Berthelot, 75005 Paris, France.
Studler
J M
JM
Burgaya
F
F
Costa
A
A
Ezan
P
P
Gelman
M
M
Girault
J A
JA
D017398:Alternative Splicing; D000818:Animals; D000906:Antibodies / Q000276:immunology; D001253:Astrocytes / Q000502:physiology; D001921:Brain / Q000201:enzymology / Q000378:metabolism; D019556:COS Cells; D004926:Escherichia coli; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D007527:Isoenzymes / Q000378:metabolism*; D009474:Neurons / Q000502:physiology; D010766:Phosphorylation; D011233:Precipitin Tests; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000378:metabolism*
D000906:Antibodies; D007527:Isoenzymes; D011994:Recombinant Proteins; D014443:Tyrosine; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D019061:src-Family Kinases