Diverse roles for the mitogen-activated protein kinase ERK2 revealed by high-throughput target identification

Abstract

Many major human oncogenes contribute to cancer in large part by activating the mitogenactivated protein kinases (MAPK) ERK1 and ERK2 (ERK). These kinases are critical in normal physiological processes from development to memory, and their activation in cancer drives growth and metastatic invasion. Understanding the effects of ERK signaling is especially important since vemurafenib, the first pharmaceutical directly targeting the MAPK pathway, has recently been approved to treat advanced melanoma. ERK controls cellular phenotypes by phosphorylating over two hundred known substrate proteins, however new ERK targets are reported frequently. We have used a chemical genetics approach to identify over one hundred novel substrates of ERK2. This approach utilizes an ERK2 kinase with "gatekeeper" mutation that allows it to bind bulky ATP analogs (AS-ERK2). AS-ERK2 can be used to label its direct substrates with thiophosphate in an in vitro kinase reaction. To achieve sufficient sensitivity we have improved on existing protocols for identification of thiophosphorylated peptides. Our improved protocol identified over one hundred novel ERK2 substrates in 3T3-L1 fibroblasts and in colon carcinoma cell lines. We investigated one novel ERK2 substrate, the transcriptional repressor ETV3, in detail and found that phosphorylation abrogates binding to DNA by ETV3, and that mutation of key phosphorylated residues to alanine blocks this effect. We also identified several thousand ETV3 targets across the genome. The wide range of genes targeted by ETV3 suggests that it may act as a key regulator of cell cycle and metabolism in some cell types. We have also identified ERK2 substrates in the DLD1 colon carcinoma cell line, including several mRNA splicing factors and members of the MLL family of histone 3 methyltransferases. We are using high-throughput sequencing and biochemical experiments to determine whether these phosphorylation sites control the function of MLL proteins. Taken together these investigations greatly expand our knowledge of the ERK signaling pathway and have revealed greater connectivity among biological processes than had been appreciated.