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 (p125(FAK)), an important substrate for tyrosine phosphorylation by other neuropeptides. NMB caused rapid increases in p125(FAK) 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 p125(FAK) tyrosine phosphorylation and other cellular processes was similar in both C6 cells and rNMB-R transfected cells. TPA (1 microM) caused 45% and the calcium ionophore, A23187, 11% of maximal tyrosine phosphorylation of p125(FAK) seen with NMB. A23187 potentiated the effect of TPA. Pretreatment with the selective PKC inhibitor, GF109203X, inhibited TPA-induced p125(FAK) 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 p125(FAK) phosphorylation either alone or with GF109203X. The tyrosine kinase inhibitor, tyrphostin A25, inhibited NMB-induced phosphorylation of p125(FAK) 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 p125(FAK) 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 p125(FAK) by 55%. These results demonstrate that activation of NMB-R can cause rapid tyrosine phosphorylation of p125(FAK). NMB-induced tyrosine phosphorylation of p125(FAK) 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 phosphorylation of p125(FAK). The ras-related small GTP-binding protein rho p21 is at least partially involved in mediating NMB-induced tyrosine phosphorylation of p125(FAK). These results suggest that similar to some other neuropeptides, activation of this pathway may be an important mechanism in mediating cellular changes by this receptor such as growth.
Tsuda
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Digestive Diseases Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892-1804, USA.
Kusui
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Jensen
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RT
D036002:ADP Ribose Transferases / Q000378:metabolism; D008841:Actin Cytoskeleton / Q000737:chemistry / Q000187:drug effects; D000199:Actins / Q000378:metabolism*; D000818:Animals; D001905:Botulinum Toxins; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D003078:Colchicine / Q000494:pharmacology; D015638:Cytochalasin D / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D019204:GTP-Binding Proteins / Q000378:metabolism*; D019886:Gastrin-Releasing Peptide / Q000378:metabolism; D007211:Indoles / Q000494:pharmacology; D008301:Maleimides / Q000494:pharmacology; D051379:Mice; D015287:Neurokinin B / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D009570:Nitriles / Q000494:pharmacology; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D018004:Receptors, Bombesin / Q000378:metabolism*; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019284:Thapsigargin / Q000494:pharmacology; D014162:Transfection / Q000235:genetics; D014407:Tumor Cells, Cultured; D010738:Type C Phospholipases / Q000378:metabolism*; D020032:Tyrphostins; D020741:rho GTP-Binding Proteins
D000199:Actins; D015815:Cell Adhesion Molecules; D004791:Enzyme Inhibitors; D007211:Indoles; D008301:Maleimides; D009570:Nitriles; D018004:Receptors, Bombesin; D020032:Tyrphostins; C079836:tyrphostin 25; D019000:Phosphotyrosine; D015638:Cytochalasin D; D000001:Calcimycin; D019284:Thapsigargin; D019886:Gastrin-Releasing Peptide; D015287:Neurokinin B; C038393:neuromedin B; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496051:Ptk2 protein, mouse; C496052:Ptk2 protein, rat; D010738:Type C Phospholipases; D001905:Botulinum Toxins; D019204:GTP-Binding Proteins; D020741:rho GTP-Binding Proteins; C070515:bisindolylmaleimide I; D013755:Tetradecanoylphorbol Acetate; D003078:Colchicine; D002118:Calcium