Comprehensive Analysis of ERK1/2 Substrates for Potential Combination Immunotherapies.

Recent clinical and therapeutic success with RAF and MEK1/2 inhibitors has revolutionized the existing treatment schemes for previously incurable cancers like melanomas. However, the overall therapeutic efficacies are still largely compromised by the dose-limiting side effects and emerging drug resistance mechanisms. Accumulating evidence has revealed the intricate nature of the RAS-RAF-MEK1/2-ERK1/2 pathway, such as activation mechanisms, kinase-substrate relationships, crosstalk with parallel signaling pathways, feedback regulations, and intimate interplay with immune responses. Limited strategies are currently available to exploit the benefits of combining RAF-MEK1/2-ERK1/2 pathway inhibitors with other targeted therapies or immunotherapies. Here, we compiled the kinase-substrate relationships and analyzed the intricate signaling networks of the renowned pathway, providing an integrated and simplified visualization, to reveal the potentials of RAS-RAF-MEK1/2-ERK1/2-based combination therapies.

Exposure to dsRNA elicits RNA interference in Brachionus manjavacas (Rotifera).

RNA interference (RNAi) is a powerful technique for functional genomics, yet no studies have reported its successful application to zooplankton. Many zooplankton, particularly microscopic metazoans of phylum Rotifera, have unique life history traits for which genetic investigation has been limited. In this paper, we report the development of RNAi methods for rotifers, with the exogenous introduction of double-stranded RNA (dsRNA) through the use of a lipofection reagent. Transfection with dsRNA for heat shock protein 90, the membrane-associated progesterone receptor, and mitogen-activated protein kinase significantly increased the proportion of non-reproductive females. Additionally, a fluorescence-based lectin binding assay confirmed the significant suppression of four of six glycosylation enzymes that were targeted with dsRNA. Suppression of mRNA transcripts was confirmed with quantitative PCR. Development of RNAi for rotifers promises to enhance the ability for assessing genetic regulation of features critical to their life history and represents a key step toward functional genomics research in zooplankton.

Gene transfer of dominant-negative mutants of extracellular signal-regulated kinase and c-Jun NH2-terminal kinase prevents neointimal formation in balloon-injured rat artery.

We previously reported that extracellular signal-regulated kinase (ERK) and c-Jun NH2-terminal kinase (JNK), belonging to mitogen-activated protein kinases, are rapidly activated in balloon-injured artery. Therefore, we examined the role of these kinase activations in neointimal formation by using an in vivo gene transfer technique. We made the dominant-negative mutants of ERK (DN-ERK) and JNK (DN-JNK) to specifically inhibit endogenous ERK and JNK activation, respectively. Before balloon injury, these mutants were transfected into rat carotid artery using the hemagglutinating virus of Japan liposome method. In vivo transfection of DN-ERK and DN-JNK significantly suppressed the activation of ERK and JNK, respectively, after balloon injury, confirming successful expression of the transfected genes. Neointimal formation at 14 and 28 days after injury was prevented by gene transfer of DN-ERK or DN-JNK. Furthermore, bromodeoxyuridine labeling index and total cell-counting analysis at 7 days showed that either DN-ERK or DN-JNK remarkably suppressed smooth muscle cell (SMC) proliferation in both the intima and the media after injury. Gene transfer of wild-type ERK (W-ERK) or JNK (W-JNK) significantly enhanced neointimal hyperplasia at 14 days after injury. Furthermore, DN-ERK and DN-JNK significantly suppressed serum-induced SMC proliferation in vitro. We obtained the first evidence that in vivo gene transfer of DN-ERK or DN-JNK prevented neointimal formation in balloon-injured artery by inhibiting SMC proliferation. Thus, ERK and JNK activation triggers SMC proliferation, leading to neointimal formation. These kinases may be the new therapeutic targets for prevention of vascular diseases.

RISK-1: a novel MAPK homologue in axoplasm that is activated and retrogradely transported after nerve injury.

Sensory neurons (SNs) of Aplysia are widely used to study the molecular correlates of learning. Among these is the activation of an Aplysia (ap) MAPK that phosphorylates the transcription factor apC/EBPbeta. Because crushing the axons of the SNs induces changes similar to learning, we tested the hypothesis that apMAPK is a point of convergence on the pathways for learning and injury. One event in common is long-term hyperexcitability (LTH), and LTH was induced in the SNs after intrasomatic injection of active vertebrate extracellular signal-regulated kinase 1 (ERK1; as an apMAPK surrogate). Nerve crush activated an axoplasmic kinase at the site of injury that phosphorylated apC/EBPbeta. Surprisingly, this was not apMAPK, but a kinase that was recognized by antibodies to vertebrate ERKs and to doubly phosphorylated, activated ERKs. The activated kinase was transported to the cell body and nucleus and its arrival was concurrent with an injury-induced increase in apC/EBPbeta mRNA and protein. We call this retrogradely transported kinase RISK-1. RISK-1 initiated the binding of apC/EBPbeta to the ERE enhancer site in vitro and an increase in ERE-binding was detected in injured neurons containing active RISK-1. Thus, Aplysia neurons contain two MAPK homologues, one of which is a late acting retrogradely transported injury signal.