Live visualization of ERK activity in the mouse blastocyst reveals lineage\specific signaling dynamics. affinity to MEK1. This low conversation 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. gene, which ends with the site of insertion (Pages et al.,?1995). The 78?bp insertion encodes 26 amino acids (Physique?3c), which are localized between Glu\340 and Pro\341 of rat ERK1. This region is Isradipine usually localized in the large loop L16 between the helices i and L16, C\terminally to the kinase domain name core (Physique?3b). We then inspected the sequence for possible similarities with other proteins, but did not find any such homology within the data base. No protein motif or phosphorylation sites were found in this this sequence. This protein was also distinct from the putative ERK1psi that was previously reported (Boulton, Nye, et al.,?1991). Thus, we clearly indicates the presence of an additional mRNA for ERK1b. This mRNA encodes for a putative protein kinase with a calculated mass of 45.6?kDa. Open in a Isradipine separate window Physique 3 Cloning of ERK1b. (a) Reverse\transcriptase Isradipine polymerase chain reaction (RT\PCR) and PCR cloning of ERK1b. Lane 1, RT\PCR with the oligonucleotide primers ERK1C850\S and ERK1\CT\AS, and total RNA from EJ cells as a template. Lane 2, RT\PCR with the oligonucleotide primers ERK1b\NT\S, ERK1\CT\AS, and total RNA from EJ cells. Lane 3, RT\PCR with the oligonucleotide primers ERK1b\CT\AS, ERK1\NT\S, and total RNA from EJ cells. Lane Isradipine 4, PCR with ERK1C850\S, ERK1\CT\AS, and ERK1 in pCDNA1. Lane 5, PCR with ERK1C850\S, ERK1\CT\AS, and ERK1b cloned into the EcoRI and XhoI sites of pCDNA1. The position of the relevant fragments and DNA markers is usually indicated. (b) Complementary DNA (cDNA) and amino acid sequences of the ERK1b\specific and some of the flanking sequence (italics). The insertion site (Glu\340 and Pro\367, instead of Pro\341 in ERK1) in ERK1 is usually indicated by squares. (c) Schematic representation of ERK1b and ERK1. The sequence of the ERK1b\specific insert and its kinase domain are indicated 3.4. Purification of the ERK1b protein To find out whether the 46\kDa ERK is indeed expressed by the mRNA cloned, we needed to purify the endogenous protein and find out if it contains the expected sequence. For this purpose, tumors of EJ cells in nude mice were excised and then lysed in a buffer containing proteinase and phosphatase inhibitors. The extract was partially fractionated to cytoplasmic and nuclear fraction and the former was subjected to anion exchange column. As described above, unlike ERK1 and ERK2, Isradipine the p46\kDa ERK did not bind the resin and was eluted in the flowthrough (Figure?4a). The 46\kDa ERK in that fraction was further purified using an affinity column consists of anti\C terminus of ERK Ab bound to agarose. The column was then extensively washed and the Rabbit Polyclonal to CXCR4 bound proteins were eluted with high pH (Figure?4b). This procedure yielded ~4000\fold purification of the 46\kDa protein that was shown to react with various anti\ERK Abs (Figure?4c) and was about 70% pure. We then further separated the p46\kDa ERK using an SDS\PAGE and the proteins.