Da. Linseman et al., A role for the small molecular weight GTPases, Rho and Cdc42, in muscarinic receptor signaling to focal adhesion kinase, J NEUROCHEM, 74(5), 2000, pp. 2010-2020
An enhanced tyrosine phosphorylation of focal adhesion kinase (FAK) is elic
ited during neuronal growth cone remodeling and requires the maintenance of
agonist-sensitive pools of phosphatidylinositol 4,5-bisphosphate (PIP2). R
ho family GTPases are putative regulators of both PIP2 synthesis and growth
cone remodeling, including neurite outgrowth elicited by muscarinic cholin
ergic receptor (mAChR) stimulation. In this study, we investigated the inte
rrelationships among Rho family GTPases, PIP2 synthesis, and mAChR signalin
g to FAK in SH-SY5Y neuroblastoma cells. Preincubation with Clostridium dif
ficile toxin B (Tox B), an inhibitor of Rho, Pac, and Cdc42, attenuated mAC
hR-stimulated FAK and paxillin tyrosine phosphorylation and lysophosphatidi
c 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 ac
tivation elicited by either phorbol ester or an mAChR agonist. In contrast,
preincubation with selective inhibitors of either Rho (C3 exoenzyme) or Rh
o kinase (HA-1077) resulted in 80-90% reductions in LPA-induced FAK phospho
rylation but only 40-50% decreases in mAChR-stimulated phosphorylation. Mor
eover, mAChR-mediated FAK phosphorylation was significantly attenuated in c
ells scrape-loaded with dominant-negative N17Cdc42 but not N17Rac1, Tox B h
ad little or no effect on agonist-sensitive pools of PIP2 but inhibited mAC
hR-driven actin cytoskeletal remodeling. The results suggest that the Rho f
amily GTPases, Rho and Cdc42, link mAChR stimulation to increases in FAK ph
osphorylation independently of effects on PIP2 synthesis.