Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1.

J.C Scimeca,T.T Nguyen,C Filloux,E Van Obberghen

doi:10.1016/s0021-9258(18)41935-7

Abstract

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.

Highlights

Using antipeptides to the C terminus of the M, 44,000 MAP kinase, ERK1, and cell extracts from unstimulated or During the last 5 years much research in the area of cell signaling has focussed on the serine/threonine MAP kinases, which are thought to play a central role in metabolic and mitogenic effects induced by various extracellular stimuli
NGF-treated PC12 cells, we obtained in addition to or phosphatase inhibitors, MAP’ kinase phosphorylation is MAP kinase itself coprecipitation of a protein with a increased on both threonine andtyrosine residues, leading to
Our observations demonstrate that the 44-kDa MAP kinase is associated, in intact PC12 cells, with a protein kinase phosphorylations

Summary

RESULTS

Coprecipitatingwith ERK1-To search for coprecipitation with ERKl of cellular activators and/or substrates,confluent. I n vitro phosphorylation was performed on eluates, and samples were analyzed by SDS-PAGE under reducing conditions (Fig. 1).In both cell types, we observed two major phosphoproteins, one in the range of 90 kDa, and another one at 44 kDa corresponding to ERK1. We found that insulin, NGF or EGF strongly stimulated the. The bandwith an estimated molecular weight of 46,000, and whose phosphorylation is significantly stimulated by insulin in HIRcells, could be detected on shorter exposures of phosphoproteins in eluates from NGF- and EGF-treatedPC12 cells. After solubilization and immunoprecipitation with anti-ERK1 antipeptides, the phosphoproteins adsorbed on the pellet were eluted by incubation with C peptide, and submitted to SDS-. We can conclude that in living PC12 cells ERKl was associated with a NGF-sensitive 90-kDa phosphoprotein

Phosphoarnino Acid Analysisof the in Vitro Phosphorylated

NO YES

PHOSPHOAMINOACID ANALYSIS

BUFFER NGF

DISCUSSION