CB1 Cannabinoid Receptors Stimulate Gβγ-GRK2-Mediated FAK Phosphorylation at Tyrosine 925 to Regulate ERK Activation Involving Neuronal Focal Adhesions.

George D Dalton,Allyn C Howlett,Jamie D Marshburn,Derek C Norford,Skyla T Carney

doi:10.3389/fncel.2020.00176

Abstract

CB1 cannabinoid receptors (CB1) are abundantly expressed in the nervous system where they regulate focal adhesion kinase (FAK) and the mitogen-activated protein kinases (MAPK) extracellular signal-regulated kinase 1 and 2 (ERK1/2). However, the role of CB1-stimulated FAK 925 tyrosine phosphorylation (Tyr-P) in regulating ERK1/2 activation remains undefined. Here, immunoblotting analyses using antibodies against FAK phospho-Tyr 925 and ERK2 phospho-Tyr 204 demonstrated CB1-stimulated FAK 925 Tyr-P and ERK2 204 Tyr-P (0–5 min) which was followed by a decline in Tyr-P (5–20 min). CB1 stimulated FAK-Grb2 association and Ras-mediated ERK2 activation. The FAK inhibitors Y11 and PF 573228 abolished FAK 925 Tyr-P and partially inhibited ERK2 204 Tyr-P. FAK 925 Tyr-P and ERK2 204 Tyr-P were adhesion-dependent, required an intact actin cytoskeleton, and were mediated by integrins, Flk-1 vascular endothelial growth factor receptors, and epidermal growth factor receptors. FAK 925 Tyr-P and ERK2 204 Tyr-P were blocked by the Gβγ inhibitor gallein, a GRK2 inhibitor, and GRK2 siRNA silencing, suggesting Gβγ and GRK2 participate in FAK-mediated ERK2 activation. Together, these studies indicate FAK 925 Tyr-P occurs concurrently with CB1-stimulated ERK2 activation and requires the actin cytoskeleton and Gi/oβγ-GRK2-mediated cross-talk between CB1, integrins, and receptor tyrosine kinases (RTKs).

Highlights

Endocannabinoid signaling in neuronal cells includes a rapid activation of focal adhesion kinase (FAK; Derkinderen et al, 2003; Dalton et al, 2013)
To determine whether Ras signaling is necessary for ERK2 activation, we examined the effect of the Ras inhibitor farnesylthiosalicylic acid (FTA) on WIN55212-2-stimulated ERK2 204 tyrosine phosphorylation (Tyr-P) in N18TG2 cells (Haklai et al, 1998)
Our pathway leading from CB1 to FAK 925 Tyr-P in the cloned N18TG2 neuronal cell line is similar to what has been observed in hippocampal slices, future studies would need to be done in hippocampal slices to examine the effects of FAK inhibition on CB1-stimulated ERK activation to determine if the mechanism is similar to what we report

Summary

Introduction

Endocannabinoid signaling in neuronal cells includes a rapid activation of focal adhesion kinase (FAK; Derkinderen et al, 2003; Dalton et al, 2013). FAK is a highly conserved non-receptor protein Tyr kinase that functions as a signal-transducing scaffold protein that regulates multiple cellular functions including proliferation, apoptosis, organization of the actin cytoskeleton, migration, CB1 Regulates ERK Via FAK and adhesion (Peng and Guan, 2011). FAK binds to the adaptor protein paxillin to dock with components of the actin cytoskeleton at focal adhesion sites (Parsons, 2003). FAK activation is regulated by Tyr-P and occurs in response to integrin engagement, as well as RTK and G protein-coupled receptor (GPCR) stimulation (Parsons, 2003; Peng and Guan, 2011; Dalton et al, 2013). Src family kinases (Src) bind to FAK phospho-Tyr 397 and phosphorylate FAK on additional Tyr residues (Tyr 407, Tyr 576/577, Tyr 861, Tyr 925) that mediate specific FAK functions such as FAK maximal activation and FAK-mediated activation of Ras signal transduction (Schlaepfer et al, 1994; Calalb et al, 1995, 1996; Schlaepfer and Hunter, 1996)

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Conclusion