Navigating the ERK1/2 MAPK Cascade.

The RAS-ERK pathway is a fundamental signaling cascade crucial for many biological processes including proliferation, cell cycle control, growth, and survival; common across all cell types. Notably, ERK1/2 are implicated in specific processes in a context-dependent manner as in stem cells and pancreatic ß-cells. Alterations in the different components of this cascade result in dysregulation of the effector kinases ERK1/2 which communicate with hundreds of substrates. Aberrant activation of the pathway contributes to a range of disorders, including cancer. This review provides an overview of the structure, activation, regulation, and mutational frequency of the different tiers of the cascade; with a particular focus on ERK1/2. We highlight the importance of scaffold proteins that contribute to kinase localization and coordinate interaction dynamics of the kinases with substrates, activators, and inhibitors. Additionally, we explore innovative therapeutic approaches emphasizing promising avenues in this field.

ASCL1-ERK1/2 Axis: ASCL1 restrains ERK1/2 via the dual specificity phosphatase DUSP6 to promote survival of a subset of neuroendocrine lung cancers.

The transcription factor achaete-scute complex homolog 1 (ASCL1) is a lineage oncogene that is central for the growth and survival of small cell lung cancers (SCLC) and neuroendocrine non-small cell lung cancers (NSCLC-NE) that express it. Targeting ASCL1, or its downstream pathways, remains a challenge. However, a potential clue to overcoming this challenage has been information that SCLC and NSCLC-NE that express ASCL1 exhibit extremely low ERK1/2 activity, and efforts to increase ERK1/2 activity lead to inhibition of SCLC growth and surival. Of course, this is in dramatic contrast to the majority of NSCLCs where high activity of the ERK pathway plays a major role in cancer pathogenesis. A major knowledge gap is defining the mechanism(s) underlying the low ERK1/2 activity in SCLC, determining if ERK1/2 activity and ASCL1 function are inter-related, and if manipulating ERK1/2 activity provides a new therapeutic strategy for SCLC. We first found that expression of ERK signaling and ASCL1 have an inverse relationship in NE lung cancers: knocking down ASCL1 in SCLCs and NE-NSCLCs increased active ERK1/2, while inhibition of residual SCLC/NSCLC-NE ERK1/2 activity with a MEK inhibitor increased ASCL1 expression. To determine the effects of ERK activity on expression of other genes, we obtained RNA-seq from ASCL1-expressing lung tumor cells treated with an ERK pathway MEK inhibitor and identified down-regulated genes (such as SPRY4, ETV5, DUSP6, SPRED1) that potentially could influence SCLC/NSCLC-NE tumor cell survival. This led us to discover that genes regulated by MEK inhibition suppress ERK activation and CHIP-seq demonstrated these are bound by ASCL1. In addition, SPRY4, DUSP6, SPRED1 are known suppressors of the ERK1/2 pathway, while ETV5 regulates DUSP6. Survival of NE lung tumors was inhibited by activation of ERK1/2 and a subset of ASCL1-high NE lung tumors expressed DUSP6. Because the dual specificity phosphatase 6 (DUSP6) is an ERK1/2-selective phosphatase that inactivates these kinases and has a pharmacologic inhibitor, we focused mechanistic studies on DUSP6. These studies showed: Inhibition of DUSP6 increased active ERK1/2, which accumulated in the nucleus; pharmacologic and genetic inhibition of DUSP6 affected proliferation and survival of ASCL1-high NE lung cancers; and that knockout of DUSP6 "cured" some SCLCs while in others resistance rapidly developed indicating a bypass mechanism was activated. Thus, our findings fill this knowledge gap and indicate that combined expression of ASCL1, DUSP6 and low phospho-ERK1/2 identify some neuroendocrine lung cancers for which DUSP6 may be a therapeutic target.

ERK2 Mutations Affect Interactions, Localization, and Dimerization.

The most frequent ERK2 (MAPK1) mutation in cancers, E322K, lies in the common docking (CD) site, which binds short motifs made up of basic and hydrophobic residues present in the activators MEK1 (MAP2K1) and MEK2 (MAP2K2), in dual specificity phosphatases (DUSPs) that inactivate the kinases, and in many of their substrates. Also, part of the CD site, but mutated less often in cancers, is the preceding aspartate (D321N). These mutants were categorized as gain of function in a sensitized melanoma system. In Drosophila developmental assays, we found that the aspartate but not the glutamate mutant caused gain-of-function phenotypes. Here, we catalogued additional properties of these mutants to accrue greater insight into their functions. A modest increase in nuclear retention of E322K was noted. Binding of ERK2 E322K and D321N to a small group of substrates and regulatory proteins was similar, in spite of differences in CD site integrity. Interactions with a second docking site, the F site, which should be more accessible in E322K, were modestly reduced rather than increased. The crystal structure of ERK2 E322K also indicated a disturbed dimer interface, and reduced dimerization was detected by a two-hybrid test; yet, it was detected in dimers in EGF-treated cells, although to a lesser extent than D321N or wt ERK2. These findings indicate a range of small differences in behaviors that may contribute to increased function of E322K in certain cancers.

Scaffold coupling: ERK activation by trans-phosphorylation across different scaffold protein species.

RAS-ERK (extracellular signal-regulated kinase) pathway signals are modulated by scaffold proteins that assemble the components of different kinase tiers into a sequential phosphorylation cascade. In the prevailing model scaffold proteins function as isolated entities, where the flux of phosphorylation events progresses downstream linearly, to achieve ERK phosphorylation. We show that different types of scaffold proteins, specifically KSR1 (kinase suppressor of Ras 1) and IQGAP1 (IQ motif-containing guanosine triphosphatase activating protein 1), can bind to each other, forming a complex whereby phosphorylation reactions occur across both species. MEK (mitogen-activated protein kinase kinase) bound to IQGAP1 can phosphorylate ERK docked at KSR1, a process that we have named "trans-phosphorylation." We also reveal that ERK trans-phosphorylation participates in KSR1-regulated adipogenesis, and it also underlies the modest cytotoxicity exhibited by KSR-directed inhibitors. Overall, we identify interactions between scaffold proteins and trans-phosphorylation as an additional level of regulation in the ERK cascade, with broad implications in signaling and the design of scaffold protein-aimed therapeutics.