The role of the ERK signalling pathway in cancer is thought to be most prominent in tumours in which mutations in the receptor tyrosine kinases RAS, BRAF, CRAF, MEK1 or MEK2 drive growth factor-independent ERK1 and ERK2 activation and thence inappropriate cell proliferation and survival. New drugs that inhibit RAF or MEK1 and MEK2 have recently been approved or are currently undergoing late-stage clinical evaluation. In this Review, we consider the ERK pathway, focusing particularly on the role of MEK1 and MEK2, the 'gatekeepers' of ERK1/2 activity. We discuss their validation as drug targets, the merits of targeting MEK1 and MEK2 versus BRAF and the mechanisms of action of different inhibitors of MEK1 and MEK2. We also consider how some of the systems-level properties (intrapathway regulatory loops and wider signalling network connections) of the ERK pathway present a challenge for the success of MEK1 and MEK2 inhibitors, discuss mechanisms of resistance to these inhibitors, and review their clinical progress.

Summary Diabetic nephropathy (DN) is the leading cause of renal failure in the world. It is characterized by albuminuria and abnormal glomerular function and is considered a hyperglycaemic “microvascular’ complication of diabetes, implying a primary defect in the endothelium. However, we have previously shown that human podocytes have robust responses to insulin. To determine whether insulin signaling in podocytes affects glomerular function in vivo we generated mice with specific deletion of the insulin receptor from their podocytes. These animals develop significant albuminuria together with histological features that recapitulate DN, but in a normoglycaemic environment. Examination of “normal” insulin responsive podocytes in vivo and in vitro demonstrates that insulin signals through the MAPK and PI3-kinase pathways via the insulin receptor and directly remodels the actin cytoskeleton of this cell. Collectively, this work reveals the critical importance of podocyte insulin sensitivity for kidney function.

Dual-specificity MAP kinase phosphatases (MKPs) provide a complex negative regulatory network that acts to shape the duration, magnitude and spatiotemporal profile of MAP kinase activities in response to both physiological and pathological stimuli. Individual MKPs may exhibit either exquisite specificity towards a single mitogen-activated protein kinase (MAPK) isoform or be able to regulate multiple MAPK pathways in a single cell or tissue. They can act as negative feedback regulators of MAPK activity, but can also provide mechanisms of crosstalk between distinct MAPK pathways and between MAPK signalling and other intracellular signalling modules. In this review, we explore the current state of knowledge with respect to the regulation of MKP expression levels and activities, the mechanisms by which individual MKPs recognize and interact with different MAPK isoforms and their role in the spatiotemporal regulation of MAPK signalling.

Although many stimuli activate extracellular signal-regulated kinases 1 and 2 (ERK1/2), the kinetics and compartmentalization of ERK1/2 signals are stimulus-dependent and dictate physiological consequences. ERKs can be inactivated by dual specificity phosphatases (DUSPs), notably the MAPK phosphatases (MKPs) and atypical DUSPs, that can both dephosphorylate and scaffold ERK1/2. Using a cell imaging model (based on knockdown of endogenous ERKs and add-back of wild-type or mutated ERK2-GFP reporters), we explored possible effects of DUSPs on responses to transient or sustained ERK2 activators (epidermal growth factor and phorbol 12,13-dibutyrate, respectively). For both stimuli, a D319N mutation (which impairs DUSP binding) increased ERK2 activity and reduced nuclear accumulation. These stimuli also increased mRNA levels for eight DUSPs. In a short inhibitory RNA screen, 12 of 16 DUSPs influenced ERK2 responses. These effects were evident among nuclear inducible MKP, cytoplasmic ERK MKP, JNK/p38 MKP, and atypical DUSP subtypes and, with the exception of the nuclear inducible MKPs, were paralleled by corresponding changes in Egr-1 luciferase activation. Simultaneous removal of all JNK/p38 MKPs or nuclear inducible MKPs revealed them as positive and negative regulators of ERK2 signaling, respectively. The effects of JNK/p38 MKP short inhibitory RNAs were not dependent on protein neosynthesis but were reversed in the presence of JNK and p38 kinase inhibitors, indicating DUSP-mediated cross-talk between MAPK pathways. Overall, our data reveal that a large number of DUSPs influence ERK2 signaling. Together with the known tissue-specific expression of DUSPs and the importance of ERK1/2 in cell regulation, our data support the potential value of DUSPs as targets for drug therapy.

Background and purpose:The ability of an agonist to induce desensitization of the m-opioid receptor (MOR) depends upon the agonist used. Furthermore, previous data suggest that the intracellular mechanisms underlying desensitization may be agonist-specific. We investigated the mechanisms underlying MOR desensitization, in adult mammalian neurons, caused by morphine (a partial agonist in this system) and DAMGO (a high-efficacy agonist). Experimental approach: MOR function was measured in locus coeruleus neurons, by using whole-cell patch-clamp electrophysiology, in rat and mouse brain slices (both wild-type and protein kinase C (PKC)a knockout mice). Specific isoforms of PKC were inhibited by using inhibitors of the receptors for activated C-kinase (RACK), and in vivo viral-mediated gene-transfer was used to transfect neurons with dominant negative mutants (DNMs) of specific G-protein-coupled receptor kinases (GRKs). Key results: Morphine-induced desensitization was attenuated by using RACK inhibitors that inhibit PKCa, but not by other isoform-specific inhibitors. Further, the PKC component of morphine-induced desensitization was absent in locus coeruleus neurons from PKCa knockout mice. The PKC-enhanced morphine-induced desensitization was not affected by over-expression of a GRK2 dominant negative mutant (GRK2 DNM). In contrast, DAMGO-induced MOR desensitization was independent of PKC activity but was reduced by over-expression of the GRK2 DNM but not by that of a GRK6 DNM. Conclusions and implications: In mature mammalian neurons, different MOR agonists can induce MOR desensitization by different mechanisms, morphine by a PKCa-mediated, heterologous mechanism and DAMGO by a GRK-mediated, homologous mechanism. These data represent functional selectivity at the level of receptor desensitization.