Detection of partially phosphorylated forms of ERK by monoclonal antibodies reveals spatial regulation of ERK activity by phosphatases

AbstractWhen cells are stimulated by mitogens, extracellular signal-regulated kinase (ERK) is activated by phosphorylation of its regulatory threonine (Thr) and tyrosine (Tyr) residues. The inactivation of ERK may occur by phosphatase-mediated removal of the phosphates from these Tyr, Thr or both residues together. In this study, antibodies that selectively recognize all combinations of phosphorylation of the regulatory Thr and Tyr residues of ERK were developed, and used to study the inactivation of ERK upon mitogenic stimulation. We found that inactivation of ERK in the early stages of mitogenic stimulation involves separate Thr and Tyr phosphatases which operate differently in the nucleus and in the cytoplasm. Thus, ERK is differentially regulated in various subcellular compartments to secure proper length and strength of activation, which eventually determine the physiological outcome of many external signals

Detection of ERK activation by a novel monoclonal antibody

AbstractThe mitogen-activated protein kinase, ERK is activated by a dual phosphorylation on threonine and tyrosine residues. Using a synthetic diphospho peptide, we have generated a monoclonal antibody directed to the active ERK. The antibody specifically identified the active doubly phosphorylated, but not the inactive mono- or non- phosphorylated forms of ERKs. A direct correlation was observed between ERK activity and the intensity in Western blot of mitogen-activated protein kinases from several species. The antibody was proven suitable for immunofluorescence staining, revealing a transient reactivity with ERKs that were translocated to the nucleus upon stimulation. In conclusion, the antibody can serve as a useful tool in the study of ERK signaling in a wide variety of organisms

Differential regulation of cumulus cell transcription during oocyte maturation in vivo and in vitro

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HAS2-AS1 is a novel LH/hCG target gene regulating HAS2 expression and enhancing cumulus cells migration

Abstract Background The cumulus expansion process is one of the LH mediated ovulatory processes. Hyaluronan synthase 2 (HAS2) regulates the synthesis of hyaluronic acid, the main component of the cumulus expansion process. Recently, the lncRNA HAS2 antisense RNA 1 (HAS2-AS1) was identified in our global transcriptome RNA-sequencing of novel ovulation associated genes. The role of HAS2-AS1 in HAS2 regulation w.as studied previously with contradictive results in different models but not in the ovary. Taken together the induction of HAS2-AS1 and the important role of HAS2 in the cumulus expansion process, we hypothesize that HAS2-AS1 regulate HAS2 expression and function in the ovary. Therefore we undertook to study the expression, regulation, and possible functional role of HAS2-AS1 in the human ovary. Results HAS2-AS1, located within the HAS2 gene that was highly regulated in our library. We found that HAS2-AS1 express mainly in cumulus cells (CCs). Furthermore, HAS2-AS1 showed low expression in immature CCs and a significant increase expression in mature CCs. Functional studies reveal that inhibition of HAS2-AS1 by siRNA caused decrease expression of HAS2. Furthermore, inhibition of HAS2-AS1 by siRNA results in decrease migration of granulosa cells. Conclusions Our results suggest that HAS2-AS1 is an LH/hCG target gene that plays a positive role in HAS2 expression and thus might play a role in regulating cumulus expansion and migration

Extracellular Signal-Regulated Kinase 1c (ERK1c), a Novel 42-Kilodalton ERK, Demonstrates Unique Modes of Regulation, Localization, and Function

Extracellular signal-regulated kinases (ERKs) are signaling molecules that regulate many cellular processes. We have previously identified an alternatively spliced 46-kDa form of ERK1 that is expressed in rats and mice and named ERK1b. Here we report that the same splicing event in humans and monkeys causes, due to sequence differences in the inserted introns, the production of an ERK isoform that migrates together with the 42-kDa ERK2. Because of the differences of this isoform from ERK1b, we named it ERK1c. We found that its expression levels are about 10% of ERK1. ERK1c seems to be expressed in a wide variety of tissues and cells. Its activation by MEKs and inactivation by phosphatases are slower than those of ERK1, which is probably the reason for its differential regulation in response to extracellular stimuli. Unlike ERK1, ERK1c undergoes monoubiquitination, which is increased with elevated cell density concomitantly with accumulation of ERK1c in the Golgi apparatus. Elevated cell density also causes enhanced Golgi fragmentation, which is facilitated by overexpression of native ERK1c and is prevented by dominant-negative ERK1c, indicating that ERK1c mediates cell density-induced Golgi fragmentation. The differential regulation of ERK1c extends the signaling specificity of MEKs after stimulation by various extracellular stimuli

miR-221/222 compensates for Skp2-mediated p27 degradation and is a primary target of cell cycle regulation by prostacyclin and cAMP.

p27(kip1) (p27) is a cdk-inhibitory protein with an important role in the proliferation of many cell types. SCF(Skp2) is the best studied regulator of p27 levels, but Skp2-mediated p27 degradation is not essential in vivo or in vitro. The molecular pathway that compensates for loss of Skp2-mediated p27 degradation has remained elusive. Here, we combine vascular injury in the mouse with genome-wide profiling to search for regulators of p27 during cell cycling in vivo. This approach, confirmed by RT-qPCR and mechanistic analysis in primary cells, identified miR-221/222 as a compensatory regulator of p27. The expression of miR221/222 is sensitive to proteasome inhibition with MG132 suggesting a link between p27 regulation by miRs and the proteasome. We then examined the roles of miR-221/222 and Skp2 in cell cycle inhibition by prostacyclin (PGI(2)), a potent cell cycle inhibitor acting through p27. PGI(2) inhibited both Skp2 and miR221/222 expression, but epistasis, ectopic expression, and time course experiments showed that miR-221/222, rather than Skp2, was the primary target of PGI(2). PGI(2) activates Gs to increase cAMP, and increasing intracellular cAMP phenocopies the effect of PGI(2) on p27, miR-221/222, and mitogenesis. We conclude that miR-221/222 compensates for loss of Skp2-mediated p27 degradation during cell cycling, contributes to proteasome-dependent G1 phase regulation of p27, and accounts for the anti-mitogenic effect of cAMP during growth inhibition