Src family kinase involvement in muscarinic receptor-induced tyrosine phosphorylation in differentiated SH-SY5Y cells.
Muscarinic receptor-mediated changes in protein tyrosine phosphorylation were examined in differentiated human neuroblastoma SH-SY5Y cells. Treatment of differentiated cells with 1 mM carbachol caused rapid increases in the tyrosine phosphorylation of focal adhesion kinase (FAK), Cas, and paxillin. The src family kinase-selective inhibitor PP1 reduced carbachol-stimulated tyrosine phosphorylation of FAK, Cas, and paxillin by 50 to 75%. In contrast, carbachol-stimulated activation of ERK1/2 was unaffected by PP1. Src family kinase activation by carbachol was further demonstrated by increased carbachol-induced tyrosine phosphorylation of the src-substrate, p120, and tyrosine phosphorylation of the src family kinase activation-associated autophosphorylation site. Site-specific FAK phosphotyrosine antibodies were used to determine that the carbachol-stimulated increase in the autophosphorylation of FAK was unaffected by pretreatment with PP1, whereas the carbachol-stimulated increase in the src family kinase-mediated phosphotyrosine of FAK was completely blocked by pretreatment with PP1. In SH-SY5Y cell lines stably overexpressing Fyn, the phosphotyrosine immunoreactivity of FAK was 625% that of control cells. Thus, muscarinic receptors activate protein tyrosine phosphorylation in differentiated cells, and the tyrosine phosphorylation of FAK, Cas, and paxillin, but not ERK1/2, is mediated by a src family tyrosine kinase activated in response to stimulation of muscarinic receptors.
Watcharasit
P
P
Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, 35294-0017, USA.
Tucholski
J
J
Jope
R S
RS
D002217:Carbachol / Q000494:pharmacology; D002454:Cell Differentiation; D029563:Cellular Apoptosis Susceptibility Protein / Q000378:metabolism; D018679:Cholinergic Agonists / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D009474:Neurons / Q000473:pathology / Q000502:physiology; D051419:Paxillin; D010641:Phenotype; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011720:Pyrazoles / Q000494:pharmacology; D011743:Pyrimidines / Q000494:pharmacology; D011976:Receptors, Muscarinic / Q000502:physiology*; D015398:Signal Transduction; D014407:Tumor Cells, Cultured / Q000473:pathology; D014443:Tyrosine / Q000378:metabolism*; D019061:src-Family Kinases / Q000502:physiology*
C108123:4-amino-5-(4-methylphenyl)-7-(tert-butyl)pyrazolo(3,4-d)pyrimidine; D029563:Cellular Apoptosis Susceptibility Protein; D018679:Cholinergic Agonists; D003598:Cytoskeletal Proteins; D004791:Enzyme Inhibitors; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011720:Pyrazoles; D011743:Pyrimidines; D011976:Receptors, Muscarinic; D014443:Tyrosine; D002217:Carbachol; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D019061:src-Family Kinases