The ERK mitogen-activated protein kinase signaling network: the final frontier in RAS signal transduction

Abstract

The RAF-MEK-ERK mitogen-activated protein kinase (MAPK) cascade is aberrantly activated in a diverse set of human cancers and the RASopathy group of genetic developmental disorders. This protein kinase cascade is one of the most intensely studied cellular signaling networks and has been frequently targeted by the pharmaceutical industry, with more than 30 inhibitors either approved or under clinical evaluation. The ERK-MAPK cascade was originally depicted as a serial and linear, unidirectional pathway that relays extracellular signals, such as mitogenic stimuli, through the cytoplasm to the nucleus. However, we now appreciate that this three-tiered protein kinase cascade is a central core of a complex network with dynamic signaling inputs and outputs and autoregulatory loops. Despite our considerable advances in understanding the ERK-MAPK network, the ability of cancer cells to adapt to the inhibition of key nodes reveals a level of complexity that remains to be fully understood. In this review, we summarize important developments in our understanding of the ERK-MAPK network and identify unresolved issues for ongoing and future study.

Keywords: PDAC; RAS GTPase; extracellular signal-regulated kinases; mitogen-activated protein kinases.

Figure 1.. ERK–MAPK signaling is a critical node in KRAS-mutant cancer.

(A) RAS regulates both the RAF–MEK–ERK and PI3K–AKT–mTORC1 signaling networks promoting cell cycle progression, cell survival, differentiation and metabolism. The mutation frequencies are indicated by % and were compiled from COSMIC v92. Gain-of-function (green text) or loss-of-function (red text) mutations are indicated. PI3Kα is activated through RAS interaction with the p110α catalytic subunit whereas EGFR activation of PI3K is through phosphorylation-dependent binding of the p85 regulatory domain. The PTEN tumor suppressor lipid phosphatase dephosphorylates PIP3, leading to inactivation of AKT. (B) Components of three-tiered ERK–MAPKKK (MAPK kinase kinase)-MAPKK (MAPK kinase)-MAPK cascade. In addition to the kinase domain, RAF kinases contain amino-terminal RAS-binding domains (RBD), where activated RAS-GTP binds to initiate a series of events that lead to activation of RAF kinase activity. The adjacent cysteine-rich domain (CRD) facilitates RAF association with membranes. A complex set of phosphorylation events regulate RAF kinase activity, with ERK feedback inhibition negative (red) and representative positive (green) phosphorylation events shown [1,4]. Total protein and kinase domain sequence identities are indicated (%/%), respectively, as determined by CLUSTALW multiple sequence alignment.

Figure 2.. ERK regulation of a complex phosphoproteome and transcriptome.

(A) ERK substrates are characterized by a S/T-P consensus phosphorylation site and two ERK binding motifs. (B) ERK substrates include protein kinases that further diversify the ERK-regulated phosphoproteome. (C) ERK regulates the expression of transcription factors through both transcriptional and posttranslational mechanisms that promote protein stability. ERK phosphorylation and activation of ELK1 which together with serum response factor (SRF) stimulates promoters containing serum response element (SRE) found in immediate early gene promoters. FOS/FRA1 and JUN heterodimers form AP-1 complexes that further stimulate genes with the TPA response element (TRE) DNA promoter consensus sequence. MYC forms heterodimers with the MAX transcription factor, which then stimulates gene transcription from E-box DNA element-containing promoters. However, MYC overexpression leads to MYC–MAX stimulation of virtually all active promoters. (D–E) Down-regulated gene expression changes following ERKi in oncogenic KRAS-driven PDAC cells. (D) The Heat map of 50 most down- and 10 most up-regulated genes at four h. after ERKi (1 μM) across seven KRAS-mutant PDAC cell lines (Pa01C, Pa14C, HPAC, PANC-1, SW1990 and HPAF-II) [10]. Heatmap was generated based on differential expression analysis of the RNA-seq data. Negative regulators of ERK are annotated with blue. (E) Top gene ontology (GO) term enrichments based on the top 100 genes up- and down-regulated at each time point in the six PDAC cell lines shown in (D). Differentially expressed genes were ranked according to the product of their scaled significance (−log10 adj. P-val.) and degree of change (log2 FC). Enrichment scores were calculated for molecular function (MF) and biological process (BP) terms [11].

Figure 3.. ERK–MAPK activity is tightly regulated in cells.

(A) Multiple ERK activation-initiated negative feedback mechanisms exist to prevent excessive ERK activation in KRAS-mutant cancers. Some involve ERK phosphorylation and inhibition of EGFR, SOS1, RAF and MEK activities. Others involve transcriptional up-regulation of genes that encode proteins that act as negative regulators of ERK signal either upstream or downstream of ERK. Red lines indicate negative feedback mechanisms. ERK phosphorylation of protein kinases or transcription factors promote a complex ERK phosphoproteome or transcriptome, respectively. (B) The Goldilocks principle is a concept based on the fairy tale ‘Goldilocks and the Three bears’ written by Robert Southey, where the protagonist of the story, Goldilocks, tasted three different bowls of porridge. She found Papa bear’s to be too hot, Mama’s to be too cold, and baby bear’s to be ‘just right’. By analogy, ERK-dependent feedback mechanisms keep the level of ERK activity ‘just right’ to drive cancer growth. Mutant RAS drives elevated and persistent ERK activity that is necessary to drive mutant KRAS-driven tumorigenic growth. However, excessive ERK activity will be deleterious to cancer cell growth, resulting in cancer cell senescence and apoptosis.