Neuromedin B Receptor Activation Causes Tyrosine Phosphorylation of p125FAK by a Phospholipase C Independent Mechanism Which Requires p21rho and Integrity of the Actin Cytoskeleton

Recent studies show that tyrosine phosphorylation by a number of neuropeptides may be an important intracellular pathway in mediating changes in cell function, particularly related to growth. Neuromedin B (NMB), a mammalian bombesin related peptide, functions through a distinct receptor, the neuromedin B receptor (NMB-R), of which little is known about its cellular basis of action. In the present study we explored the ability of NMB-R activation to cause tyrosine phosphorylation of focal adhesion kinase (p125FAK), an important substrate for tyrosine phosphorylation by other neuropeptides. NMB caused rapid increases in p125FAK phosphorylation which reached maximum at 2 min in both rat C6 glioblastoma cells which possess native NMB-Rs and rat neuromedin B receptor (rNMR-R) transfected BALB 3T3 cells. NMB had a half-maximal effect was at 0.4 nM and was 30-fold more potent than gastrin-releasing peptide (GRP). The stoichiometric relationships between increased p125FAK tyrosine phosphorylation and other cellular processes was similar in both C6 cells and rNMB-R transfected cells. TPA (1 μM) caused 45% and the calcium ionophore, A23187, 11% of maximal tyrosine phosphorylation of p125FAK seen with NMB. A23187 potentiated the effect of TPA. Pretreatment with the selective PKC inhibitor, GF109203X, inhibited TPA-induced p125FAK tyrosine phosphorylation, but it had no effect on the NMB stimulation. Pretreatment with thapsigargin completely inhibited NMB-stimulated increases in [Ca2+]i, but had no effect on NMB-stimulation of p125FAK phosphorylation either alone or with GF109203X. The tyrosine kinase inhibitor, tyrphostin A25, inhibited NMB-induced phosphorylation of p125FAK by 52%. However, tyrphostin A25 did not inhibit NMB-stimulated increases in [3H]inositol phosphates. Cytochalasin D, an agent which disrupts actin microfilaments, inhibited BN- and TPA-induced tyrosine phosphorylation of p125FAK completely. In contrast, colchicine, an agent which disrupts microtubules, had no effect. Pretreatment with Clostridium botulinum C3 exoenzyme which inactivates the small GTP-binding protein rho p21, also inhibited tyrosine phosphorylation of p125FAK by 55%. These results demonstrate that activation of NMB-R can cause rapid tyrosine phosphorylation of p125FAK. NMB-induced tyrosine phosphorylation of p125FAK is independent of NMB-induced changes in [Ca2+]i or PKC. The integrity of the actin cytoskeleton but not of microtubules is necessary for NMB-stimulated phosphorylatio