Specific interactions of neuronal focal adhesion kinase isoforms with Src kinases and amphiphysin.
Focal adhesion kinase (FAK) is a non-receptor tyrosine kinase that activates Src family kinases via SH2- and SH3-mediated interactions. Specific FAK isoforms (FAK+), responsive to depolarization and neurotransmitters, are enriched in neurons. We analyzed the interactions of endogenous FAK+ and recombinant FAK+ isoforms containing amino acid insertions (boxes 6,7,28) with an array of SH3 domains and the c-Src SH2/SH3 domain tandem. Endogenous FAK+ bound specifically to the SH3 domains of c-Src (but not n-Src), Fyn, Yes, phosphtidylinositol-3 kinase, amphiphysin II, amphiphysin I, phospholipase Cgamma and NH2-terminal Grb2. The inclusion of boxes 6,7 was associated with a significant decrease in the binding of FAK+ to the c-Src and Fyn SH3 domains, and a significant increase in the binding to the Src SH2 domain, as a consequence of the higher phosphorylation of Tyr-397. The novel interaction with the amphiphysin SH3 domain, involving the COOH-terminal proline-rich region of FAK, was confirmed by coimmunoprecipitation of the two proteins and a closely similar response to stimuli affecting the actin cytoskeleton. Moreover, an impairment of endocytosis was observed in synaptosomes after internalization of a proline-rich peptide corresponding to the site of interaction. The data account for the different subcellular distribution of FAK and Src kinases and the specific regulation of the transduction pathways linked to FAK activation in the brain and implicate FAK in the regulation of membrane trafficking in nerve terminals.
Messina
Samantha
S
Department of Experimental Medicine, Section of Human Physiology, University of Genova, Italy.
Onofri
Franco
F
Bongiorno-Borbone
Lucilla
L
Giovedì
Silvia
S
Valtorta
Flavia
F
Girault
Jean-Antoine
JA
Benfenati
Fabio
F
D000818:Animals; D001923:Brain Chemistry; D019556:COS Cells; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D015151:Immunoblotting; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000378:metabolism*; D009474:Neurons / Q000201:enzymology / Q000378:metabolism*; D011233:Precipitin Tests; D011485:Protein Binding / Q000502:physiology; D020033:Protein Isoforms / Q000737:chemistry / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000737:chemistry / Q000378:metabolism*; D051381:Rats; D013574:Synaptosomes / Q000737:chemistry / Q000378:metabolism; D018909:src Homology Domains / Q000502:physiology; D019061:src-Family Kinases / Q000737:chemistry / Q000378:metabolism*
D009419:Nerve Tissue Proteins; D020033:Protein Isoforms; C075637:amphiphysin; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496052:Ptk2 protein, rat; D019061:src-Family Kinases