Elsevier

Cellular Signalling

Volume 25, Issue 8, August 2013, Pages 1665-1677
Cellular Signalling

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

https://doi.org/10.1016/j.cellsig.2013.03.020Get rights and content

Highlights

  • A novel pathway triggering CB1-stimulated FAK activity was elucidated in neurons.

  • CB1 stimulated FAK 576/577 tyrosine phosphorylation in a triphasic manner.

  • CB1-stimulated FAK 576/577 tyrosine phosphorylation was adhesion-dependent.

  • Maximal FAK activation required cooperative signaling between integrins and RTKs.

  • Maximal FAK activation involved tyrosine phosphatase-mediated Src activation.

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 that 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 that 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 and 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.

Introduction

CB1 receptors are predominantly expressed in the nervous system and mediate many of the neuronal effects produced by the major psychoactive component of Cannabis sativa Δ9-THC, the endocannabinoids anandamide and 2-arachidonoylglycerol (2-AG), and synthetic cannabinoid drugs (e.g., CP55940 and WIN55212-2) (see [1] for review). CB1 is a G protein-coupled receptor (GPCR) that associates with pertussis toxin-sensitive Gi/o proteins to regulate a variety of signal transduction pathways including inhibition of adenylyl cyclase, inhibition of L-, N-, and P/Q-type Ca2 + channels, induction of immediate early gene expression, stimulation of nitric oxide production, activation of members of the mitogen-activated protein kinase (MAPK) family, and activation of FAK [1], [2]. FAK is a ubiquitously expressed nonreceptor protein tyrosine kinase that localizes to multi-protein complexes found at the cell membrane called focal adhesions (FAs) in which integrins link the actin cytoskeleton to proteins of the extracellular matrix (ECM) [3]. Activated FAK mediates many of the downstream signaling events emanating from FAs that regulate cell proliferation, survival, migration, and adhesion [3], [4]. FAK activation occurs through Tyr-P and begins with FAK phosphorylation at Tyr 397 which creates a high affinity binding site for Src that then phosphorylates FAK on five additional Tyr residues (Tyr 407, Tyr 576/577, Tyr 861, and Tyr 925) [5], [6], [7]. Tyr 576/577 are located in the activation loop of the FAK central catalytic domain and their phosphorylation is required for maximal FAK catalytic activity. Studies have shed minimal light on the cellular mechanisms that regulate CB1-mediated FAK activation which appears to involve integrin activation, PKA inhibition, and Src activation [8], [9], [10]. During development of the central nervous system, endocannabinoid signaling networks regulate proliferation, migration, specification, and survival of neural progenitors [11], [12]. Given the crucial role of FAK in these biological processes, it is important to gain a better understanding of the cellular and molecular mechanisms that regulate CB1-FAK signaling pathways in neuronal cells [4].

The aim of the present study was to investigate the signaling pathways that regulate CB1-stimulated maximal FAK catalytic activation in neuronal N18TG2 cells that express endogenous CB1 receptors. To accomplish this, immunoblotting analyses were conducted using phosphorylation site-specific antibodies against FAK Tyr 576/577 and Tyr 397. Our results revealed the time-course of CB1-mediated FAK 397 and 576/577 Tyr-P are markedly different in N18TG2 cells. FAK 576/577 Tyr-P occurred in three phases: Phase I (0–2 min) involved maximal Tyr-P, Phase II (5–20 min) involved a rapid decline in Tyr-P, and Phase III (> 20 min) involved a plateau in Tyr-P at submaximal levels. In contrast, FAK 397 Tyr-P was monophasic and significantly lower in magnitude. CB1-mediated FAK 397 Tyr-P and Phase I FAK 576/577 Tyr-P involved protein tyrosine phosphatase (PTP1B and Shp1/Shp2)-mediated Src activation, PKA inhibition, integrin activation, and were adhesion-dependent. Phase I FAK 576/577 Tyr-P also involved cooperative signaling between RTKs (Flk-1 VEGFRs and EGFRs) and integrins. These studies have identified a novel cellular mechanism by which CB1 induces maximal FAK enzymatic activity in neuronal cells that involves crosstalk between CB1, RTKs, and integrins.

Section snippets

Materials

Reagents were purchased from Sigma Chemical Company (St. Louis, MO, USA), unless otherwise stated. CP55940 ((—)-cis-3R-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4R-3(3-hydroxypropyl)-1R cyclohexanol) and SR141716A (N-(piperidin-1yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-H-pyrazole-3-carboxamide) were provided by the National Institute of Drug Abuse drug supply program. Methanandamide [(R)-(+)-Arachidonyl-1′-hydroxy-2′-propylamide], 2-AG, WIN55212-2, and tetrahydrolipstatin

The time-course of CB1-stimulated FAK phosphorylation at tyrosines 397 and 576/577 differs in N18TG2 cells

Kinetic analysis revealed the time-course and magnitude of CB1-stimulated FAK phosphorylation at Tyr 397 differs from Tyr 576/577 in N18TG2 cells (Fig. 1). CB1-stimulated FAK 576/577 Tyr-P was time-dependent and occurred in three phases. The synthetic CB1 agonist WIN55212-2 (0.01 μM) produced a robust and transient increase in FAK 576/577 Tyr-P that reached maximal levels in 1 to 2 min (Phase I), declined to a near basal level by 20 min (Phase II), and plateaued at submaximal levels after 20 min

Discussion

Tyr 397 is the autophosphorylation site of FAK and is involved in FAK initial activation [3]. Phosphorylated Tyr 397 binds Src which phosphorylates Tyr 576/577 in the FAK activation loop to produce maximal FAK catalytic activity [3]. Our data suggest that CB1-mediated FAK 397 and 576/577 Tyr-P are differentially regulated. CB1-stimulated FAK 397 Tyr-P is monophasic and significantly lower in magnitude than Phase I FAK 576/577 Tyr-P, although both involved integrin activation, PKA inhibition,

Conclusions

The information gained regarding the cellular mechanisms of CB1-stimulated maximal FAK activation demonstrates how protein kinases, protein phosphatases, and different classes of cell membrane receptors play a role in the complex signaling networks that regulate cellular function. We determined that CB1 stimulates maximal FAK catalytic activity in neuronal cells via integrin activation, PKA inhibition, as well as protein tyrosine phosphatase-mediated Src activation. Integrins and Src stimulate

Acknowledgments

This work was supported by NIH grants R01-DA03690 to ACH and F32-DA026295 to GDD. GDD was an RJR-Leon Golberg Post-doctoral Scholar in Pharmacology at Wake Forest University. The authors declare no conflicts of interest.

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