title: 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 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.
authors.affiliation: Department of Physiology and Pharmacology, Wake Forest University School of Medicine, Winston-Salem, NC 27157, USA. gedalton@wakehealth.edu
authors.forename: George D
authors.initials: GD
authors.lastname: Dalton
authors.forename: Lynda J
authors.initials: LJ
authors.lastname: Peterson
authors.forename: Allyn C
authors.initials: AC
authors.lastname: Howlett
mesh_terms: D000818:Animals; D048588:Benzoxazines / Q000494:pharmacology; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000037:antagonists & inhibitors / Q000378:metabolism; D005353:Fibronectins / Q000494:pharmacology; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D016023:Integrins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D007797:Laminin / Q000494:pharmacology; D051379:Mice; D009025:Morpholines / Q000494:pharmacology; D009281:Naphthalenes / Q000494:pharmacology; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000037:antagonists & inhibitors / Q000378:metabolism; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11 / Q000037:antagonists & inhibitors / Q000378:metabolism; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6 / Q000037:antagonists & inhibitors / Q000378:metabolism; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D043884:Receptor, Cannabinoid, CB1 / Q000819:agonists / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects; D013997:Time Factors; D040301:Vascular Endothelial Growth Factor Receptor-2 / Q000037:antagonists & inhibitors / Q000378:metabolism; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
chemical_list: D048588:Benzoxazines; D005353:Fibronectins; D016023:Integrins; D007797:Laminin; D009025:Morpholines; D009281:Naphthalenes; D009842:Oligopeptides; D034741:RNA, Small Interfering; D043884:Receptor, Cannabinoid, CB1; C070417:(3R)-((2,3-dihydro-5-methyl-3-((4-morpholinyl)methyl)pyrrolo-(1,2,3-de)-1,4-benzoxazin-6-yl)(1-naphthalenyl))methanone; C047981:arginyl-glycyl-aspartic acid; D037342:Pertussis Toxin; D066246:ErbB Receptors; D040301:Vascular Endothelial Growth Factor Receptor-2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019061:src-Family Kinases; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D053824:Protein Tyrosine Phosphatase, Non-Receptor Type 6; C516609:Ptpn1 protein, mouse