Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1.
Microtubule-associated protein (MAP) kinases form a group of serine/threonine kinases stimulated by various growth factors such as nerve growth factor (NGF) and hormones such as insulin. Interestingly, MAP kinases are thought to participate in a protein kinase cascade leading to cell growth as they have been shown to phosphorylate and activate ribosomal protein S6 kinase. To further evaluate the interactions between the different components of this cascade, we looked at the possible coprecipitation of MAP kinase activator(s) or MAP kinase substrate(s) with MAP kinase. Using antipeptides to the C terminus of the M(r) 44,000 MAP kinase, ERK1, and cell extracts from unstimulated or NGF-treated PC12 cells, we obtained in addition to MAP kinase itself coprecipitation of a protein with a M(r) in the 90,000 range. We further show that this protein is a protein kinase since it becomes phosphorylated on serine residues, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to a polyvinylidene difluoride membrane. In vitro phosphorylation performed before sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates NGF-sensitive phosphorylation of this 90-kDa protein on both serine and threonine; the serine phosphorylation is likely to be due to autophosphorylation, and the threonine phosphorylation due to phosphorylation by the copurifying MAP kinase. Furthermore, immunoprecipitation of this 90-kDa protein was obtained with antibodies to S6 kinase II. Finally, using in situ chemical cross-linking, we were able to demonstrate in intact cells the occurrence of an anti-ERK1 immunoreactive species with a molecular mass of approximately 125,000 compatible with a complex between ERK1 and a 90-kDa S6 kinase. Taken together, our observations demonstrate that the 44-kDa MAP kinase is associated, in intact PC12 cells, with a protein kinase which is very likely to be S6 kinase II. In conclusion, our data represent strong evidence for a physiological role of the MAP kinase-S6 kinase cascade in PC12 cells. Finally, our antipeptides provide us with a powerful tool to search for additional physiologically relevant substrates for MAP kinase, a key integrator enzyme for growth factors and hormones.
Scimeca
J C
JC
Institut National de la Santé et de la Recherche Médicale U145, Faculté de Médecine, Nice, France.
Nguyen
T T
TT
Filloux
C
C
Van Obberghen
E
E
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000906:Antibodies; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D015151:Immunoblotting; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016716:PC12 Cells; D010455:Peptides / Q000138:chemical synthesis / Q000276:immunology; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000302:isolation & purification / Q000378:metabolism*; D019893:Ribosomal Protein S6 Kinases; D013388:Succinimides / Q000494:pharmacology
D000596:Amino Acids; D000906:Antibodies; D010455:Peptides; D010750:Phosphoproteins; D013388:Succinimides; D011494:Protein Kinases; D019893:Ribosomal Protein S6 Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; C019358:disuccinimidyl suberate