CB1 cannabinoid receptors promote maximal FAK catalytic activity by stimulating cooperative signaling between receptor tyrosine kinases and integrins in neuronal cells

Abstract

Tyrosine phosphorylation (Tyr-P) of focal adhesion kinase (FAK) regulates FAK activation. Phosphorylated FAK Tyr 397 binds Src family kinases (Src), which in turn directly phosphorylate FAK Tyr 576/577 to produce maximal FAK enzymatic activity. CB1 cannabinoid receptors (CB1) are abundantly expressed in the nervous system and influence FAK activation by presently unknown mechanisms. The current investigation determined CB1-stimulated maximal FAK catalytic activity is mediated by Gi/o proteins in N18TG2 neuronal cells, and that G12/13 regulation of Rac1 and RhoA occurs concomitantly. Immunoblotting analyses using antibodies against FAK phospho-Tyr 397 and phospho-Tyr 576/577 demonstrated the time-course of CB1-stimulated FAK 576/577 Tyr-P occurred in three phases: Phase I (0-2 min) maximal Tyr-P, Phase II (5-20 min) rapid decline in Tyr-P, and Phase III (>20 min) plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B, Shp1/Shp2)-mediated Src activation, Protein Kinase A (PKA) inhibition, and integrin activation. Phase I maximal FAK 576/577 Tyr-P also required cooperative signaling between receptor tyrosine kinases (RTKs) and integrins. The integrin antagonist RGDS peptide, Flk-1 vascular endothelial growth factor receptor (VEGFR) antagonist SU5416, and epidermal growth factor receptor (EGFR) antagonist AG 1478 blocked Phase I FAK 576/577 Tyr-P. CB1 agonists failed to stimulate FAK Tyr-P in the absence of integrin activation upon suspension in serum-free culture media. In contrast, cells grown on the integrin ligands fibronectin and laminin displayed increased FAK 576/577 Tyr-P that was augmented by CB1 agonists and blocked by the Src inhibitor PP2 and Flk-1 VEGFR antagonist SU5416. Taken together, these studies have identified a complex integrative pathway utilized by CB1 to stimulate maximal FAK 576/577 Tyr-P in neuronal cells.

Atypical Endocannabinoid Signaling Initiates a New Form of Memory-Related Plasticity at a Cortical Input to Hippocampus

“…This raises the possibility that the CB 1 R promotes lppLTP via actions on actin regulatory signaling, an idea in alignment with evidence that CB 1 R initiates actin reorganization in dissociated cells (Roland et al 2014;Njoo et al 2015) and rapidly (~2 min) activates both FAK and the small GTPase RhoA in N18TG2 neuroblastoma cells (Dalton et al 2013). FAK is a non-receptor tyrosine kinase that mediates integrin effects on the actin cytoskeleton throughout the body (Fabry et al 2011).…”

“…One possibility is that electophysiological stimulation of the LPP engages elements that are not downstream from CB 1 R activation but nonetheless are required for shifting LPP terminals into the enhanced release state. We tested if integrin class adhesion proteins, which co-operate with CB 1 R in actin regulatory signaling in cultured cells (Dalton et al 2013), fill this critical role. Integrins are dimeric transmembrane adhesion receptors for extracellular matrix and cell surface proteins that are expressed throughout the brain by neurons and glia (Park and Goda 2016).…”

CB1 Cannabinoid Receptors Couple to Focal Adhesion Kinase to Control Insulin Release

“…A rise of intracellular Ca 2ϩ being a prerequisite of insulin release is also compatible with FAK and FAK-related kinase (76,77) activation and cytoskeletal rearrangements during the second phase of insulin secretion, particularly because vesicle docking during exocytosis is reliant on the Ca 2ϩ -driven assembly of the SNARE machinery. Exocytosis involves a series of highly coordinated and sequential steps.…”

“…FAK is an appealing target because it can suppress Rho GTPase activity, thereby promoting FA turnover (35). Considering that CB 1 Rs can modulate the activity of RhoA (and other small GTPases) (34,76), FAK is poised to orchestrate cystoskeletal remodeling, particularly FA plaque assembly, required for insulin release. CB 1 R activation establishes maximal FAK activity upon cooperative signaling with cell surface tyrosine kinase receptors and the sequential phosphorylation of Tyr-397 and Tyr-576/577 FAK residues (76).…”

“…Considering that CB 1 Rs can modulate the activity of RhoA (and other small GTPases) (34,76), FAK is poised to orchestrate cystoskeletal remodeling, particularly FA plaque assembly, required for insulin release. CB 1 R activation establishes maximal FAK activity upon cooperative signaling with cell surface tyrosine kinase receptors and the sequential phosphorylation of Tyr-397 and Tyr-576/577 FAK residues (76). Here, we showed FAK phosphorylation at Tyr-397, which is considered a permissive step for the recruitment of Src-family kinases, whose second-phase phosphorylation of an additional five Tyr residues in the activation loop of the kinase, confers maximal FAK activity (76).…”