ERK protein kinase was isolated and identified in the early 1990s as a signal transduction protein that transmits mitogen signals. It is often found in the cytosol and is rapidly translocated into the nucleus of the cell when it is activated by mitogens such as various growth factors. The phosphoric acid ERK1/2 acts on transcription factors such as c-Jun, NF-AT, and NF-κB to exert biological effects. ERK is the first MAPK member to be discovered. It is the most widely studied and plays an important role in the development of tumors. The ERK-associated intracellular signaling pathway is thought to be the classical MAPK pathway, an important part of a series of kinase enzymatic cascades that transduce extracellular stimuli from the cell membrane into the nucleus.
ERK is a silk/threonine protein kinase belonging to the members of Mitogen-Activated Protein Kinases (MAPKs). ERK is an important transduction protein that transmits mitogen signals in the body and is expressed in most cells. The current study found that ERK includes several subfamilies of ERK1/ERK2, ERK3/ERK4, and ERK5. Among them, ERK1/ERK2 is the earliest cloned member of the MAPKs family, collectively referred to as ERK1/2, with relative molecular masses of 42 kd and 44 kd, respectively. ERK1/2 is primarily activated by mitogen stimuli and subsequently leads to activation of a range of transcription factors that ultimately regulate cell proliferation and differentiation. ERK1/2 has up to 90% homology and is a proline-directed serine/threonine kinase. Like other MAPKs family kinases, activation of ERK1/2 requires the phosphorylation of serine (Y) and threonine (T) sites to be active.
The RAS/RAF/MEK/ERK pathway is the most mature in the MAPK pathway and plays an important role in the cell signaling regulatory network. ERK is an important member of this pathway, and the activation process of the ERK cascade has been confirmed. Under external stimulation, Ras protein is activated by cell membrane enrichment, Raf protein is activated by Ras phosphorylation, and then its C-terminal catalytic domain binds to MAPK kinase (MEK), further phosphorylating and activating MEK1/2. MEK1/2 has dual specificity and is activated by phosphorylation of two regulatory sites of threonine and tyrosine at the active site of ERK protein. Activated ERK transfers to the nucleus and phosphorylates transcription factors (CREB), cytoskeleton-associated protein (CAP), and enzymes (RSK) to regulate related gene expression. In addition, it is involved in various physiological processes such as cell growth, development, division, migration, metabolism, and apoptosis.
Figure 1. The RAS/RAF/MEK/MAPK pathways
ERK includes two subtypes with up to 90% homology: ERK1 and ERK2. Although the two subtype sequences are similar, they all have their own independent functions, and ERK2 can well compensate for the functional defects of ERK1. For example, mice that are knocked out ERK2 cause embryo death. Like many kinases, ERK contains a long chain of polypeptides whose N-terminus and C-terminus are mutually curled to form a typical bilobal structure. When MEK activates ERK, phosphorylation of its N-terminal Thr183 and Tyr185 sites results in a conformational change near the N-terminus and ATP-binding site. This effect is transmitted to the C-terminus such that the C-terminal active site binds to the substrate protein to phosphorylate its serine/threonine residue. The special property of ERK diphosphorylation is determined by a cyclic structure called Loop 12 in its molecule. The ring is located on the surface of the molecule and adjacent to the active site, some of which form a lip-like structure called the "phosphorylated lip." This region is considered to be a key structure that determines the activity of ERK protein kinases.
In neuronal apoptosis, the ERK pathway has a dual role. Although most studies have demonstrated that the ERK pathway has an anti-apoptotic effect in neurons, ERK signaling-induced pro-apoptotic effects have also been observed. The different effects of the ERK pathway may result from the different types of neurons, stimuli, interactions with other MAPK pathways used in these studies, and factors that may not be known.
ERK promotes proliferation of tumor cells
The tumor is a disorder of cell cycle regulation, and abnormal regulation of cell cycle makes uncontrolled proliferation of tumor cells the most basic biological feature of malignant tumors. Cyclin D1 is a key cell cycle regulatory protein that promotes cell transition from G1 to S phase. Cyclin D1 is expressed in normal tissues with low or no expression but is often overexpressed in various tumor tissues. When various stimuli activate ERK, activated ERK can rapidly phosphorylate some early gene-encoded proteins (c-Jun, c-Fos) and activate multiple transcription factors to induce CyclinD1 expression. D1 activates E2F1 (E2F transcription factor 1) via the Cyclin D/CDK4/CKI/6-pRbE2F pathway, which promotes cell cycle progression from the G1 phase to the S phase, thereby promoting cell division and proliferation. The activated E2F1 can be activated by phosphorylation of ERK induced by positive feedback, further accelerating cell division and proliferation. Overexpression of Cyclin D1 plays an important role in the development of tumors. Corona et al. used a MEK inhibitor PD98059 in colon cancer cells to inhibit ERK phosphorylation and found that D1 expression was inhibited, thereby inhibiting tumor cell proliferation, indicating that ERK1/2 is important in cell cycle G1 to S phase transition. The antisense oligonucleotide Cyclin D1 inhibits the activity of Cyclin D1, which in turn inhibits the growth of tumor cells. This provides a new target for clinical treatment of tumors. McCubrey et al. found Ras mutations in various malignant tumors such as melanoma, ovarian cancer, and colon cancer, which led to abnormal activation of downstream MEK and ERK and caused abnormal proliferation of tumor cells.
Activated ERK1/2 also inhibits p27 expression. The p27 protein can prevent cells from inhibiting cell proliferation through G1/S phase transformation. Activated ERK1/2 can inhibit cell cycle arrest by inhibiting p27 expression, thereby promoting cell proliferation. Studies have shown that in gastric cancer, breast cancer, liver cancer and other cancer tissues, the expression of ERK1/2 and p27 is negatively correlated, and the activation of ERK1/2 can down-regulate the expression of p27 protein, thereby promoting tumor cell proliferation.
Ki-67 is a nuclear antigen expressed in proliferating cells and is a marker of nuclear proliferation. The study found that the expression of Ki-67 is related to the cell cycle, which is not expressed in G0 phase, begins to express in G1 phase, gradually increases in S phase and G2 phase, peaks in M phase, and disappears rapidly after mitosis. Studies have shown that in a variety of cancer tissues, the expression of the proliferation marker Ki-67 is positively correlated with the expression of P-ERK1/2, both of which are highly expressed and over-activated by the ERK pathway. It indicated that the abnormal activation of the ERK pathway can promote the expression of nuclear proliferation related gene Ki-67, thereby promoting the proliferation of tumor cells.
ERK inhibits apoptosis of tumor cells
ERK also exerts an anti-apoptotic effect by inhibiting the expression of the pro-apoptotic proteins Bim and Bad. MEK inhibitors can be used in combination with Bcl-2 family inhibitors to induce apoptosis, and this synergistic effect is mediated by the proapoptotic factor Bim phosphorylation. Studies have also shown that the ERK signaling pathway can directly or indirectly inhibit the activity of caspase-3, an apoptosis-inducing effector, thereby blocking various apoptosis-induced apoptosis processes. MEK inhibitors in melanoma cells can activate pro-apoptotic proteins, promote the mitochondrial release of apoptosis-inducing factors, activate caspase-3, and promote apoptosis. Activated ERK enhances cyclin E expression and inactivates p27 Kip1 cell cycle inhibitor protein, which regulates apoptosis in several ways.
Figure 2. Current targeting strategies of the RAS–ERK signaling pathway
The RAS-RAF-MEK-ERK signaling pathway is over-activated in tumors, most commonly due to activating mutations in the KRAS, NRAS and BRAF genes. Recently, the use of compounds that target ERK signaling, such as RAF or MEK inhibitors, has contributed to significant clinical improvement in metastatic melanoma and promising clinical activity in other tumor types. However, response rates are highly variable and the efficacy of these drugs is primarily limited by the development of drug resistance.
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