Large-Scale Discovery of ERK2 Substrates Identifies ERK-Mediated Transcriptional Regulation by ETV3

Abstract

The mitogen-activated protein kinase (MAPK) ERK2 is ubiquitously expressed in mammalian tissues and is involved in a wide range of biological processes. Although MAPKs have been intensely studied, identification of their substrates remains challenging. We have optimized a chemical genetic system using analog-sensitive ERK2,a form of ERK2 engineered to utilize an analog of ATP, to tag and isolate ERK2 substrates in vitro. This approach identified 80 proteins phosphorylated by ERK2, 13 of which are known ERK2 substrates. The 80 substrates are associated with diverse cellular processes, including regulation of transcription and translation, and mRNA processing, as well as regulation of the activity of the Rho-family guanosine triphosphatases. We found that one of the newly identified substrates, ETV3 (a member of the E-twenty six family of transcriptional regulators) was extensively phosphorylated on sites within canonical and non-canonical ERK motifs. Phosphorylation of ETV3 regulated transcription by preventing its binding to DNA at promoters for several thousand genes, including some involved in negative feedback regulation of itself and of upstream signals.

Identification of Extracellular Signal-regulated Kinase 1 (ERK1) Direct Substrates using Stable Isotope Labeled Kinase Assay-Linked Phosphoproteomics

“…The cellular components analysis indicated that ERK1 substrates fell into four major categories (Fig. 5A), cytoplasm, nucleus, membrane, and cytoskeleton, a distribution consistent with a previous report (40). In comparison with total human proteome (28,612 proteins) and phosphoproteome (11,479 proteins) having GO annotations from Uniprot and PhosphoSitePlus, respectively, the proportions of cytoplasm, nucleus, and cytoskeleton were significantly increased (Fig.…”

“…4C). We further compared our phosphorylation motif and D domain enrichment with other methods previously reported for in vitro MAPK substrate screening, including ASKA for ERK2 substrates (40) and high throughput in vitro kinase assay for p38 (34). Although three studies shared common features for the consensus motif SP/TP (supplemental Fig.…”

“…Recently Thibault and colleagues further expanded the ERK1-dependent repertoire, which contains 155 proteins with 232 phosphorylation sites using a rat cell line (44). In addition, two previously mentioned studies identified in vivo substrates of ERK2 (40) and p38 (34), which also belong to mitogen activation protein kinase (MAPK) family. Overlapping our data with each of those studies was provided in supplemental Fig.…”

Gain-of-function Mutations in Transient Receptor Potential C6 (TRPC6) Activate Extracellular Signal-regulated Kinases 1/2 (ERK1/2)

“…Numerous proteins and signaling pathways can influence this cascade through input at its various levels, with activation of Raf by the small GTPase Ras representing the classical pathway. Similarly, the output can be wildly disparate, with over 160 substrates for ERK1 and -2 identified (45)(46)(47) as capable of affecting diverse responses (46). The mechanisms whereby the pathway is able to execute specific responses to different stimuli remain incompletely understood, although the use of scaffolding proteins and distinct pools of kinases in different subcellular locales appears critical (48).…”

Empirical inference of circuitry and plasticity in a kinase signaling network

“…The maturation of phosphoproteomics techniques based on mass spectrometry (MS) is now allowing the simultaneous quantification of several thousands of phosphorylation sites per experiment, and approaches to derive kinase activity from these large-scale phosphoproteomics datasets have been reported (11)(12)(13)(14). One such approach, named kinase substrate enrichment analysis (KSEA), is based on the premise that, because each phosphorylation site is the result of a kinase's catalytic activity, phosphoproteomic profiling provides a means by which to capture and measure the activities of all kinases expressed in the system under investigation (14).…”