A role for the small molecular weight GTPases, Rho and Cdc42, in muscarinic receptor signaling to focal adhesion kinase.
An enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) is elicited during neuronal growth cone remodeling and requires the maintenance of agonist-sensitive pools of phosphatidylinositol 4,5-bisphosphate (PIP2). Rho family GTPases are putative regulators of both PIP2 synthesis and growth cone remodeling, including neurite outgrowth elicited by muscarinic cholinergic receptor (mAChR) stimulation. In this study, we investigated the interrelationships among Rho family GTPases, PIP2 synthesis, and mAChR signaling to FAK in SH-SY5Y neuroblastoma cells. Preincubation with Clostridium difficile toxin B (Tox B), an inhibitor of Rho, Rac, and Cdc42, attenuated mAChR-stimulated FAK and paxillin tyrosine phosphorylation and lysophosphatidic acid (LPA)-induced FAK phosphorylation to a similar extent (75% decreases at 200 pg/ml Tox B) but did not affect mitogen-activated protein kinase activation elicited by either phorbol ester or an mAChR agonist. In contrast, preincubation with selective inhibitors of either Rho (C3 exoenzyme) or Rho kinase (HA-1 077) resulted in 80-90% reductions in LPA-induced FAK phosphorylation but only 40-50% decreases in mAChR-stimulated phosphorylation. Moreover, mAChR-mediated FAK phosphorylation was significantly attenuated in cells scrape-loaded with dominant-negative N17Cdc42 but not N17Rac1. Tox B had little or no effect on agonist-sensitive pools of PIP2 but inhibited mAChR-driven actin cytoskeletal remodeling. The results suggest that the Rho family GTPases, Rho and Cdc42, link mAChR stimulation to increases in FAK phosphorylation independently of effects on PIP2 synthesis.
Linseman
D A
DA
Department of Pharmacology, Mental Health Research Institute, University of Michigan, Ann Arbor, USA.
Hofmann
F
F
Fisher
S K
SK
D000199:Actins / Q000502:physiology; D001905:Botulinum Toxins / Q000494:pharmacology; D019274:Botulinum Toxins, Type A; D003598:Cytoskeletal Proteins / Q000378:metabolism; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids / Q000494:pharmacology; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D018721:Muscarinic Agonists / Q000494:pharmacology; D051419:Paxillin; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D010716:Phosphatidylinositols / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000502:physiology*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D014407:Tumor Cells, Cultured; D020764:cdc42 GTP-Binding Protein / Q000502:physiology*; D020741:rho GTP-Binding Proteins / Q000037:antagonists & inhibitors / Q000502:physiology*; D054460:rho-Associated Kinases
D000199:Actins; D003598:Cytoskeletal Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008246:Lysophospholipids; D018721:Muscarinic Agonists; C496061:PXN protein, human; D051419:Paxillin; D019269:Phosphatidylinositol 4,5-Diphosphate; D010716:Phosphatidylinositols; D010750:Phosphoproteins; D011976:Receptors, Muscarinic; C096323:rimabotulinumtoxinB; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D020928:Mitogen-Activated Protein Kinases; D001905:Botulinum Toxins; D019274:Botulinum Toxins, Type A; D020764:cdc42 GTP-Binding Protein; D020741:rho GTP-Binding Proteins