Identification of a c-Jun N-terminal kinase-2-dependent signal amplification cascade that regulates c-Myc levels in ras transformation.

Ras is one of the most frequently activated oncogenes in cancer. Two mitogen-activated protein kinases (MAPKs) are important for ras transformation: extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase 2 (JNK2). Here we present a downstream signal amplification cascade that is critical for ras transformation in murine embryonic fibroblasts. This cascade is coordinated by ERK and JNK2 MAPKs, whose Ras-mediated activation leads to the enhanced levels of three oncogenic transcription factors, namely, c-Myc, activating transcription factor 2 (ATF2) and ATF3, all of which are essential for ras transformation. Previous studies show that ERK-mediated serine 62 phosphorylation protects c-Myc from proteasomal degradation. ERK is, however, not alone sufficient to stabilize c-Myc but requires the cooperation of cancerous inhibitor of protein phosphatase 2A (CIP2A), an oncogene that counteracts protein phosphatase 2A-mediated dephosphorylation of c-Myc. Here we show that JNK2 regulates Cip2a transcription via ATF2. ATF2 and c-Myc cooperate to activate the transcription of ATF3. Remarkably, not only ectopic JNK2, but also ectopic ATF2, CIP2A, c-Myc and ATF3 are sufficient to rescue the defective ras transformation of JNK2-deficient cells. Thus, these data identify the key signal converging point of JNK2 and ERK pathways and underline the central role of CIP2A in ras transformation.

Thapsigargin-sensitive Ca(2+)-ATPases account for Ca2+ uptake to inositol 1,4,5-trisphosphate-sensitive and caffeine-sensitive Ca2+ stores in adrenal chromaffin cells.

In chromaffin cells of adrenal medulla, heterogeneity of Ca2+ stores has been suggested with respect to the mechanisms of Ca2+ uptake and release. We have examined Ca(2+)-ATPases responsible for loading of Ca2+ stores in these cells for their sensitivity to thapsigargin, a highly selective inhibitor of the SERCA [sarco(endo)plasmic reticulum calcium ATPase] family of intracellular Ca2+ pumps. Using immunostaining, we studied the distribution of Ca(2+)-ATPases, and of receptors for inositol 1,4,5-trisphosphate (InsP3) and ryanodine, in the density-gradient fractions of microsomes from bovine adrenal medulla. In parallel, we examined distribution profiles of ATP-dependent Ca2+ uptake in the same fractions, along with subcellular markers for plasma membranes and endoplasmic reticulum (ER). Two Ca(2+)-ATPase-like proteins (116 and 100 kDa) were detected, consistent with the presence of SERCA 2b and SERCA 3 isoenzymes of Ca2+ pumps. The distribution of these putative Ca(2+)-ATPase iso-enzymes paralleled that of InsP3 and ryanodine receptors. This distribution of ER Ca(2+)-ATPases, as determined immunologically, was consistent with that of thapsigargin-sensitive, but not of thapsigargin-insensitive, ATP-dependent Ca2+ uptake. In contrast, the distribution profile of the thapsigargin-insensitive Ca2+ uptake was strongly correlated to that of plasma membranes, and co-distributed with plasma membrane Ca(2+)-ATPase detected immunologically. In isolated, permeabilized chromaffin cells, InsP3 and caffeine induced Ca2+ release following an ATP-dependent Ca2+ accumulation to the stores. This accumulation was abolished by thapsigargin. Together, these data strongly indicate that the thapsigargin-sensitive, presumably SERCA-type Ca(2+)-ATPases account for Ca2+ uptake to InsP3-sensitive, as well as to caffeine-sensitive, Ca2+ stores in bovine adrenal chromaffin cells.

Isolation of chromaffin cell thapsigargin-sensitive Ca2+ store in light microsomes from bovine adrenal medulla.

1. A subcellular fractionation procedure for bovine adrenal glands was designed with the aim to study the biochemical properties of Ca2+ stores in chromaffin cells. 2. The thapsigargin-sensitive compartment of Ca2+ stores was found to be highly enriched in a light microsomal fraction (LMF) on a 15-30% linear sucrose gradient, and was found to be essentially devoid of contamination by plasma, mitochondrial or secretory granule membranes. 3. A Ca(2+)-pumping ATPase was identified in this LMF as a 97 kDa protein forming an acid-stable, Ca(2+)-dependent, thapsigargin-sensitive phosphorylated intermediate upon incubation with [gamma-32P]ATP, suggesting this protein to represent a SERCA-3 isoform of Ca2+ ATPases. 4. A major 162 kDa protein, previously demonstrated in the isolated chromaffin cells, was enriched in the LMF, distributing on sucrose gradients in parallel with the thapsigargin-sensitive Ca2+ uptake. 5. LMF appears to represent a part of the thapsigargin-sensitive Ca2+ store of chromaffin cells, and should be useful for further studies of the store properties at the subcellular and molecular level.