In our chemical genetics screen, we have focused on identifying substrates of ERK2 due to its integral role in development and differentiation, as well as its role in cancers carrying activating mutations in B-Raf, K-Ras, EGFR and other tyrosine kinases. Through a combination of chemical genetics, metabolic labeling (see below) and analysis of multiple biological replicates by quantitative mass spectrometry, we were able to identify 80 ERK2 substrate proteins, including 67 novel substrates of this well-studied kinase. Perhaps the most striking aspect of the 67 novel substrates identified in our screen was the diversity of biological processes that they represent, which includes motor proteins, metabolic regulators, cell cycle and apoptosis proteins and proteins associated with nuclear structure, DNA organization, ubiquitination and cytoskeletal organization, among many others. Interestingly, even within this diverse group, many substrates cluster around a few biological processes; these clusters may provide a new perspective on how signaling networks exert control over complicated behaviors.
As one instance of multiple novel ERK2 substrates clustering around a particular biological function, we identified four novel substrates involved in microtubule regulation and mitosis. ERK1/2 activity has been shown to be necessary for the G2
/M transition in Xenopus oocytes,13
and it has been associated with progression through early G2
and with G2
/M transition in mammalian cells. A precise understanding of the timing and role of ERK activity in G2
/M remains elusive, with the overall effect of ERK-pathway inhibition being dependent on cell type, timing (chronic vs. acute) and the mechanism of inhibition.14
In contrast, ERK1/2 activity has a clearly established role in mediating genomic instability as a result of oncogenic mutations in upstream Ras or Raf signaling proteins.14-16
Several mechanisms by which ERK1/2 could influence mitosis have been suggested, including activation of mitotic phosphatases Cdc25B and Cdc25C in both Xenopus and mammalian cells17,18
and regulation of microtubule and spindle assembly.19
Several high-throughput screens have also shown that activation of K-Ras in colon carcinoma cells sensitizes them to loss of mitotic protein including Polo-like kinase 1 (PLK1) and the microtubule-associated proteins survivin and targeting protein for Xenopus kinesin-like protein 2 (TPX2).20-22
In our study, new targets identified using AS-ERK2 include TPX2, DLG7, CEP170 and GTSE1, all of which are involved in microtubule assembly during mitosis.23-26
These, along with previously reported microtubule-associated substrates, such as components of the dynein complex, suggest a more integral role for ERK2 in coordinating mitosis through regulation of microtubule assembly and bundling. Molecular functions for these phosphorylation events remain to be determined, but it is intriguing that one signaling pathway seems to impinge on the mitotic apparatus at so many points. The collection of mitotic ERK2 substrates may represent potential targets in carcinoma cells carrying activated MAPK pathway that could be exploited for therapeutic effect, perhaps in combination with upstream (e.g., Raf or MEK) inhibitors.
AS-ERK2 substrates also cluster around the regulators and effector proteins of the Rho family of small GTPases, which are broadly involved in cytoskeleton dynamics and cell motility.27
ERK1/2 has been shown to localize to focal adhesions, and its activity is involved in signaling from focal adhesions to Rho and its immediate target ROCK as well as interacting with focal adhesion kinase (FAK) to control disassembly of focal adhesions (reviewed in ref. 28
). However, the mechanisms by which ERK signaling regulates Rho-family guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs) are not well-understood. Novel AS-ERK2 substrates include the Rho-family GEFs DOCK1 and ARHGEF17, the Rho GAP myosin IxB (MYO9B) and the Rho-family effector proteins CDC42EP1 and CDC42EP2. It is worth noting that ERK1/2-dependent phosphorylation has also been previously observed on GEF-H1 and ARHGAP26, both of which regulate RhoA.29,30
These multiple related ERK 1/2 substrates suggest that crosstalk between ERK signaling and the Rho GTPases could occur at several points, both up- and downstream from the Rho GTPases themselves. It will be interesting to determine the functional significance of these phosphorylation sites on related proteins in this network: does phosphorylation on any site lead to a small change, with the ensemble phosphorylation then driving a much larger overall effect, or is phosphorylation of one of these sites sufficient to induce a significant phenotypic effect, with the multiple different sites then representing a fail-safe mechanism? Mutation of these phosphorylation sites to phospho-mimetic or non-phosphorylatable residues under different stimulation conditions will be needed to parse out their individual or ensemble effects.
In sum, the set of substrates identified using AS-ERK2 provides an abundance of interconnections among otherwise distinct signaling processes. Novel substrates connecting ERK2 to apparently unrelated pathways include GLI2, which may represent a mechanism for crosstalk with the Hedgehog signaling pathway, GRB10-interacting GYF protein 2 (TNRC15), which is involved in activity of the insulin receptor, the spicing factor RNA binding protein 9 (FOX2) and the microRNA-regulating protein 5'-3' exoribonuclease 2 (XRN2). Parsing how these pathways work together to coordinate cellular behaviors will require detailed characterization of molecular functions and interactions as well as careful analysis of phenotypes in a wide range of biological systems.