ERK1b, a 46‐kDa ERK isoform that is differentially regulated by MEK

Abstract

The extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) are members of the mitogen-activated protein kinase family. Using various stimulated rodent cells and kinase activation techniques, we identified a 46-kDa ERK. The kinetics of activation of this ERK isoform was similar to that of ERK1 and ERK2 under most but not all circumstances. We purified this isoform from rat cells followed by its cloning.The sequence of this isoform revealed that it is an alternatively spliced version of the 44-kDa ERK1 and therefore we termed it ERK1b. Interestingly, this isoform had a 26-amino acid insertion between residues 340 and 341 of ERK1, which results from Intron 7 insertion to the sequence. Examining the expression pattern, we found that ERK1b is detected mainly in rat and particularly in Ras-transformed Rat1 cells. In this cell line, ERK1b was more sensitive to extracellular stimulation than ERK1 and ERK2.Moreover, unlike ERK1 and ERK2, ERK1b had a very low binding affinity to MEK1. This low interaction led to nuclear localization of this isoform when expressed together with MEK1 under conditions in which ERK1 and ERK2 are retained in the cytoplasm. In addition, ERK1b was not coimmunoprecipitated with MEK1.We identified a new, 46-kDa ERK alternatively spliced isoform. Our results indicate that this isoform is the major one to respond to exogenous stimulation in Ras-transformed cells, probably due to its differential regulation by MAPK/ERK kinase and by phosphatases.

The ERK Signaling Cascade Inhibits Gonadotropin-Stimulated Steroidogenesis

“…Interestingly, the basal activity of ERK1b in rLHR-4 cells was as high as that of ERK1, only modestly increased 5-20 min after stimulation (~2-fold), and declined to basal level 40 min later. The kinetics, which are different from that of ERK1 and ERK2, suggests a differential mode of ERK1b-regulation as recently demonstrated in EJ cells [ 19 ]. We next examined LH, which like hCG, specifically acts via the LH/CG-receptors.…”

“…Serum-starved rLHR-4 cells were stimulated with hCG, which signals via the LH receptor [ 2 ], and phosphorylation of the activation TEY motif of ERK was then assessed using a Western blot analysis with DP-ERK Ab [ 24 ]. Considerable staining of three bands at 42, 44 and 46 kDa (ERK2, ERK1 and ERK1b respectively [ 19 ]) was detected in the resting, non-stimulated, cells. The intensity of staining of ERK2 and ERK1 was enhanced (~5-fold) 5-20 min after the addition of hCG, and remained high (~3-fold) up to 60 min after stimulation.…”

“…Subconfluent cultures were serum-starved for 16 h, and subsequently incubated for selected time intervals with the indicated agents in the presence or absence of various inhibitors. Following stimulation, cells were washed twice with ice-cold phosphate buffered saline, once with Buffer A (50 mM -glycerophosphate, pH 7.3, 1.5 mM EGTA, 1 mM EDTA, 1 mM DTT, and 0.1 mM sodium vanadate, [ 19 ]), and were subsequently harvested in ice-cold Buffer A + proteinase inhibitors [ 19 ]. Cell lysates were centrifuged at (20,000xg, 20 min).…”

“…The supernatant was assayed for protein content and subjected to a Western blot analysis or to immunoprecipitation as below. For the detection of StAR protein, cells were lysed in RIPA Buffer [ 19 ] and subjected to Western blot analysis.…”

“…During the final step of immunoprecipitation, pellets were washed with buffer A, resuspended in 15 μl of buffer A, and incubated (20 min, 30°C) with 5 μl of 2 mg/ml myelin basic protein (MBP) and 10 μl of 3x reaction mix (30 mM MgCl2, 4.5 mM DTT, 75 mM β-glycerophosphate, pH 7.3, 0.15 mM Na3VO4, 3.75 mM EGTA, 30 μM calmidazolium, 2.5 mg/ml bovine serum albumin and 100 μM [γ 32 P]-ATP (2 cpm/fmol)). The phosphorylation reactions were terminated by addition of sample buffer and boiling (5 min) and the samples were analyzed by SDS-PAGE and autoradiography as previously described [ 19 ].…”