Derkinderen P, Toutant M, Kadaré G, Ledent C, Parmentier M, Girault J A
INSERM U536, Institut du Fer à Moulin, 17 Rue du Fer à Moulin, 75005 Paris, France.
J Biol Chem. 2001 Oct 12;276(41):38289-96. doi: 10.1074/jbc.M105630200. Epub 2001 Jul 23.
In hippocampus endocannabinoids modulate synaptic function and plasticity and increase tyrosine phosphorylation of several proteins, including focal adhesion kinase (FAK). Autophosphorylation of FAK on Tyr-397 is generally a critical step for its activation, allowing the recruitment of Src family kinases, and phosphorylation of FAK and associated proteins. We have examined the mechanisms of the regulation of FAK by cannabinoids in rat and mouse hippocampal slices. Anandamide and 2-arachidonoylglycerol, two endocannabinoids, and Delta9-tetrahydrocannabinol, stimulated tyrosine phosphorylation of FAK+6,7, a neuronal splice isoform of FAK, on several residues including Tyr-397. Cannabinoids increased phosphorylation of p130-Cas, a protein associated with FAK, but had no effect on PYK2, a tyrosine kinase related to FAK and enriched in hippocampus. Pharmacological experiments and the use of knockout mice demonstrated that the effects of cannabinoids were mediated through CB1 receptors. These effects were sensitive to manipulation of cAMP-dependent protein kinase, suggesting that they were mediated by inhibition of a cAMP pathway. PP2, an Src family kinase inhibitor, prevented the effects of cannabinoids on p130-Cas and on FAK+6,7 tyrosines 577 and 925, but not 397, indicating that FAK autophosphorylation was upstream of Src family kinases in response to CB1-R stimulation. Endocannabinoids increased the association of Fyn, but not Src, with FAK+6,7. In hippocampal slices from Fyn -/- mice, the levels of p130-Cas were increased, and the effects of endocannabinoids on tyrosine phosphorylation, including of Tyr-397, were completely abolished. These results demonstrate the specific functional association of Fyn with FAK+6,7 in a pathway regulated by endocannabinoids, in which Fyn may play roles dependent and independent of its catalytic activity.
在海马体中，内源性大麻素可调节突触功能和可塑性，并增加包括粘着斑激酶（FAK）在内的多种蛋白质的酪氨酸磷酸化。FAK在酪氨酸397位点的自磷酸化通常是其激活的关键步骤，可促使Src家族激酶的募集以及FAK和相关蛋白质的磷酸化。我们研究了大鼠和小鼠海马切片中大麻素对FAK的调节机制。两种内源性大麻素花生四烯乙醇胺和2-花生四烯酸甘油酯，以及Δ9-四氢大麻酚，可刺激FAK+6,7（FAK的一种神经元剪接异构体）在包括酪氨酸397在内的多个位点的酪氨酸磷酸化。大麻素增加了与FAK相关的蛋白质p130-Cas的磷酸化，但对与FAK相关且在海马体中富集的酪氨酸激酶PYK2没有影响。药理学实验和基因敲除小鼠的使用表明，大麻素的作用是通过CB1受体介导的。这些作用对cAMP依赖性蛋白激酶的操作敏感，表明它们是由cAMP途径的抑制介导的。Src家族激酶抑制剂PP2可阻止大麻素对p130-Cas以及FAK+6,7酪氨酸577和925位点的作用，但不能阻止对酪氨酸397位点的作用，这表明在对CB1-R刺激的反应中，FAK自磷酸化位于Src家族激酶的上游。内源性大麻素增加了Fyn与FAK+6,7的结合，但未增加Src与FAK+6,7的结合。在Fyn基因敲除小鼠的海马切片中，p130-Cas的水平升高，内源性大麻素对酪氨酸磷酸化（包括酪氨酸397位点）的作用完全消失。这些结果表明，在由内源性大麻素调节的途径中，Fyn与FAK+6,7存在特定的功能关联，其中Fyn可能发挥与其催化活性相关和不相关作用。
