Tyrosine phosphorylation of a mitogen-activated protein kinase-like protein occurs at a late step in exocytosis. Studies with tyrosine phosphatase inhibitors and various secretagogues in rat RBL-2H3 cells.
Several inhibitors of tyrosine phosphatases, which included vanadate/H2O2, phenylarsine oxide, and diamide, blocked exocytosis in basophilic RBL-2H3 cells that had been transfected with the gene for the muscarinic m1 receptor. Because this block was observed whether the secretagogue acted through receptors (i.e. antigen and the muscarinic agonist, carbachol) or by direct activation of intracellular mechanisms (i.e. A23187, A23187 in combination with phorbol 12-myristate 13-acetate, and thapsigargin), the inhibitors appeared to act at a step distal to the mobilization of Ca2+ and activation of protein kinase C. All secretagogues caused the tyrosine phosphorylation of a 40-kDa protein, whereas the inhibitors caused a hyperphosphorylation of this protein. Therefore, both tyrosine kinase and phosphatase activities appear to regulate this phosphorylation which may, in turn, regulate secretion. The 40-kDa protein was identified as a mitogen-activated protein kinase-like protein on the basis of its reactivity to anti-mitogen-activated protein kinase antibodies. In addition, when cells were stimulated the tyrosine phosphorylated and the immunoreactive protein comigrated as a doublet on one-dimensional and as multiple phosphorylated forms on two-dimensional gel-electrophoretic systems.
Santini
F
F
Laboratory of Chemical Pharmacology, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, Maryland 20892.
Beaven
M A
MA
D000199:Actins / Q000378:metabolism; D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002217:Carbachol / Q000494:pharmacology; D002460:Cell Line; D004591:Electrophoresis, Polyacrylamide Gel; D005089:Exocytosis; D006596:Hexosaminidases / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D007700:Kinetics; D015471:Leukemia, Basophilic, Acute; D010743:Phospholipids / Q000378:metabolism; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D019000:Phosphotyrosine; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors*; D051381:Rats; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000031:analogs & derivatives / Q000378:metabolism; D014638:Vanadates / Q000494:pharmacology
D000199:Actins; D007295:Inositol Phosphates; D010743:Phospholipids; D010750:Phosphoproteins; D019000:Phosphotyrosine; D016718:Arachidonic Acid; D000001:Calcimycin; D014638:Vanadates; D014443:Tyrosine; D002217:Carbachol; D006861:Hydrogen Peroxide; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D017027:Protein Tyrosine Phosphatases; D006596:Hexosaminidases; D002118:Calcium