Extracellular Signal-Regulated Kinase 1 (ERK1)
Created by Karen Brown
The protein kinase superfamily is comprised of a multitude of proteins, including extracellular signal-regulated kinase 1 (ERK1) found in homo sapiens. ERK1 is a serine-threonine transferase involved in the Ras-dependent MAP kinase cascade that initiates and regulates cell proliferation and differentiation as well as the cell cycle progression. The function of ERK1 is significant in that it is directly involved in regulation of the cell growth cycle. The transition of the G1 phase of the cell cycle into S phase occurs with prolonged activation of ERK1. Activity of ERK1 is associated with pro-proliferative signals and the termination of transcription genes that down regulate proliferation. In addition, ERK1 also triggers the expression of the cyclin-dependent kinase (cdk) inhibitor p21, which prevents progression of the cell cycle at G1 phase. It has also been suggested by experimental study that ERK1 is involved in regulating the progression from the G2 phase of the cell cycle into mitosis. However, the significance of the role of ERK1 in this aspect of the cell cycle continues to be a topic of investigation.1 ERK1 is also involved in cytoskeletal formation through phosphorylation of microtubule-associated protein 2 (MAP2), which regulates microtubule growth.2 Lack of proper function of ERK1 and subsequent alteration of the kinase pathway has been linked to overtranscription of DNA, hyperproliferation of cells, and carcinogenesis. ERK1 is thus one of the many focuses of study in the investigation of the mechanisms of cancer.
Yeast (saccharomyces cerevisiae) protein FUS3 (PDB = 2b9f) bears structural homology in its non-phosphorylated form with ERK1, as seen in the superposition of this protein with ERK1. FUS3 is involved in a MAP kinase pathway that regulates mating activity in yeast cells. FUS3 functions to regulate cell cycle progression by activating FAR1, which mediates cell cycle arrest and has a binding motif specific to FUS3 protein. FUS3 is inactivated by the phosphatase MSG4 and is involved in cellular structure through phosphorylation of the scaffold protein STE5.1
The classification of proteins categorizes ERK1 and FUS3 as transferases. ERK1 is composed of identical A and B chains to form the polymeric structure and consists of 382 amino acid residues (mw = 88078.80 daltons) 3. The secondary structure of subunit A is identical to the B monomer and is characterized by 34% α-helical structure (18 α-helices) and 12% β sheet structure (15 β strands). Conversely, the non-phosphorylated form of FUS3 is comprised of a single A chain and consists of 353 amino acid residues (mw = 41278.91 daltons),5 with secondary structure bearing 40% α-helical character (18 α-helices) and 13% β sheet character (11 β strands). Through protein sequence analysis, chain A of non-phosphorylated FUS3 shows homology with ERK1, with a query coverage of 88%, an e-value of 4x10-119 (<0.01 %) and a total and maximum score of 349.6 Comparison of the amino acid sequences of ERK1 and non-phosphorylated FUS3 shows multiple sequence alignments, with a pair wise score of 46.0, indicating a 46% agreement between residues when compared to total alignment length. 7
Structural components of ERK1 include ionic coordination with sodium ion (Na+) at the aspartic acid residue at position 184, with the other ligand chemical components of ERK1 being (2R,3R,4S,5R)-2-(4-amino-5-iodo-7H-pyrrolo([2,3-D]pyrimidin-7-yl)-5-(hydroxymethyl)tetrahydrofuran-3,4-diol and sulfate ion, SO4.1 A hinge region is located between β5 and β6 strands which connects the N-lobe and the C-lobe of the molecule. The N-lobe is predominantly composed of β strands, whereas the C-lobe is rich in α helices. A backside binding site is located in the β1/β2 region, along with a D-motif binding site in the β6/αD region, an FXFP site in the αG region and an ATP binding site located in the β8 stretch. The activation loop containing the phosphorylation site of ERK1 is located between the β8 and αF regions of the protein. Activation of ERK1 occurs upon dual phosphorylation at the TXY motif in this activation loop, specifically at residues Thr202 and Tyr-204. The upstream kinases bind at the D-motif binding site, the FXFP binding site or the Backside binding site. The D-motif binding site contains the L16 loop (residues 332-353) as well as the αC-β4 loop, the D-helix, the E-helix and the β7-β8 turn in the C-terminal region.8 The D-motif binding site interacts with upstream kinases as well as MAP kinase phosphatases, scaffolding proteins and substrates of ERK1 through recognition of the hydrophobic and basic amino acid residues on these molecules at the R/K2,3-X1-6-ΦA-X-ΦB (Φ indicating hydrophobiticy) consensus sequence.9
Through study of crystalline structure of mono-phosphorylated ERK1, a change in conformation was noted following mono-phosphorylation at Tyr204, with a further conformational change upon subsequent phosphorylation at Thr202. This suggests that the inactive form and the basal active form differ in conformation from the fully activated kinase form, which is characterized by an extended conformation. Dual phosphorylation at the TXY activation site results in a conformation change that removes the Val-205 side chain from the reaction site, moving the Ser and Thr residues into the site. Concerning the C-helix in the mono-phosphorylated form of ERK1, it has been speculated that a hydrogen bond between the C-helix at Arg87 and the phosphorylated Thr202 residue results in the formation of a salt bridge between Lys-71 in the β3 strand and Glu88 in the C-helix. This salt bridge occurs in the fully activated form of ERK1 and is necessary for catalytic activity.8 The catalytic site of the ERK1 amino acid chain is located in residues 166 through 171 in the DLKPSN motif, with D, K, S and N serving as the catalytic residues. This site consists of alpha helical secondary structure.10
Substrate recognition following activation of ERK1 involves the most commonly the Pro-Xaa-Ser/Thr-Pro amino acid sequence.11 Molecules that are subject to phosphorylation by ERK1 include the transcription activator Elk1 that promotes gene transcription and the ribosomal s6 kinase P90RSK downstream from ERK1, which regulates transcription factors. ERK1 also phosphorylates the translation repressor protein eukaryotic translation initiation factor 4E binding protein 1 (1E1F4EBP1), microtuble-associated protein 2 (MAP2), heat shock transcription factor 4 (HSF4) and the transcription factor spermatogenic leucine zipper 1 (SPZ1). Phosphorylation by ERK1 occurs at KSP motifs and is dependent upon Magnesium (Mg2+), which acts as an inorganic cofactor. 12
Inhibition of ERK1 occurs with dephosphorylation at one or both phosphorylation sites in the active site, with dephosphorylation at a single phosphorylation adequate to induce inactivity. Dephosphorylation occurs via protein Ser/Thr phosphatases, protein Tyr phosphatases and dual-specificity phosphatases, (MKPs). These phosphatases act independently of the upstream MEK kinase proteins.11 ERK1 is inactivated upon dephosphorylation at Tyr-204 by the protein tyrosine phosphatase tumor suppressor receptor-type tyrosine-protein phosphatase eta (PTPRJ).12 Binding of MEK N-terminal peptide at the D-motif binding site has also been shown to downregulate activity of ERK1, suggesting that study of this complex may further research with regard to ERK inhibitors. In this inhibition, as well as in interaction with phosphatase peptides, the activation loop of ERK1 changes conformation such that the FXFP binding site is exposed. This exposure of the FXFP binding site permits the recognition of the F/Y-X-F/Y-R amino acid sequence on the ERK1 substrates Elk1, Egr1, and c-Fos.8
ERK1 functions in complexes with the proto-oncogene B-Raf, HRAS1, MAP2K1, MAPK3, and RGS14, which functions in regulation of G-protein signaling. Of significance is the binding that occurs between ERK1 and the viral protein HIV-1 Nef, which function in viral replication and inhibits ERK1 kinase function. ERK1 is also found to interact with beta-arrestin 2 (ARRB2), which regulates G-protein coupled receptor activity. Transmembrane glycoprotein ADAM15 also interacts with ERK1 in a phosphorylation-dependent mechanism.12
The ERK1 protein structure discussed here is with reference to the wild type form, with isoforms of ERK1 (ERK1b, ERK1c, ERK1d) existing in small minority due to alternative splicing. These isoforms are activated through isoforms of the MEK kinases which also result due to alternative splicing.11 The ERK type protein exists in two major wild type isoforms: ERK1 and ERK2. Although ERK1 and ERK2 isoforms of the ERK protein have structural similarity, these two isoforms serve different biological functions. Due to determined differences in the D-motif binding site, the FXFP binding site and the backside binding site of these isoforms, these sites can be potentially exploited in order to produce isoform-specific inhibitors.11
The study of ERK1 continues to be a topic of study in the field of science. The various functional roles of ERK1 - the role of ERK1 in cell differentiation, cell reproduction, and cytoskeleton formation, as well as its involvement in the kinase cascade - lend signficiance to this protein in multiple contexts. Further study of this protein has thus multiple advantages, with the possibility of the discovery of novel information not only with respect to the scientific study of cells but also to the behavior and treatment of cancer.