Autophosphorylation of Tyr(397) 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 spli cing. Two exons code for additional peptides of six and seven residues ('bo xes' 6 and 7), located on either side of Tyr(397), which increase its autop hosphorylation. Using in situ hybridization and a monoclonal antibody (Mab7 7) which does not recognize FAK containing box 7, we show that, although mR NAs 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 isofo rms fused to green fluorescent protein were all targeted to focal adhesions in nonneuronal 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) in-dependently and additively, whereas the y had a weak effect on FAK kinase activity towards poly(Glu,Tyr). Src-famil y kinases were also able to phosphorylate Tyr(397) in cells, but this phosp horylation was decreased in the presence of box 6 or 7, and abolished in th e presence of both. Thus the additional exons characteristic of neuronal is oforms of FAK do not alter its targeting, but change dramatically the phosp horylation of Tyr(397). They increase its autophosphorylation in vitro and in transfected COS-7 cells, whereas they prevent its phosphorylation when c otransfected with Src-family kinases.