Extracellular Signal-regulated Kinase Substrate Research Articles

Abstract 5677: βTrCP1 is a substrate of extracellular signal-regulated kinase 2

Abstract ERK1 and ERK2, downstream kinases of MEKs, transduce activation signals to a large number of cytosolic and nuclear substrates specifically involved in cellular signaling of transcription factors responsible for particular cellular processes. Thus, dysregulation of the cascade frequently leads to the development of diverse diseases, including > 90% of all cancers. However, although signaling mechanisms of ERKs by phosphorylation have well-known, long-term protein amount regulation has not clearly elucidated. In this study, we proved that βTrCP1 is a novel binding protein of ERK2. The interaction between ERK2 and βTrCP1 was occurred via F-box and linker domains spanning aa 192-265. Amino acid alignment of βTrCP1 showed that there are two independent putative sites for ERK docking. The mutations of the putative docking sites from lysine to alanine abrogated the interaction between ERK2 and βTrCP1. Importantly, ERK activation induced by epidermal growth factor induces βTrCP1 phosphorylation, resulting in facilitation of βTrCP1 half-life. Although the phosphorylation sites of βTrCP1 by ERK2 has not been identified, βTrCP1 tumor suppressive activity may be regulated via ERK2 when the cells are stimulated by tumor promoters such as EGF. Citation Format: Ga-Eun Lee, Cheol-Jung Lee, Hyun-Jung An, Weidong Chen, Dohyun Jeung, Youngwon Choi, Yong-Yeon Cho. βTrCP1 is a substrate of extracellular signal-regulated kinase 2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5677.

Interaction of compounds derived from the Chinese medicinal formula Huangqi Guizhi Wuwu Tang with stroke-related numbness and weakness targets: An in-silico docking and molecular dynamics study

Huangqi Guizhi Wuwu Tang (HGWT) is a traditional Chinese herbal formula used for managing post-stroke symptoms. Existing research have supported the use of this formula particularly for stroke-related numbness and weakness (SRNW); however, their mechanisms of actions are not fully understood. This study aims to investigate the molecular mechanisms of components from HGWT targeting specific proteins related to numbness and weakness through computational docking and molecular dynamics (MD) simulations. A total of 786 compounds from HGWT were retrieved from a herbal compound database and docked against a candidate SRNW target protein, with the asernestioside B (HQ068)-mitogen-activated protein kinase 3 (MAPK3) complex predicted to exhibit the highest binding affinity (−10.4 kcal/mol) and number of ligand-receptor contacts. Subsequent molecular dynamics (MD) simulations were performed in triplicate on the apo-MAPK3 protein and asernestioside B -bound form in a solvated system for 200 ns per trajectory to ascertain the stability of the enzyme-ligand complex, and to determine the structural impact of ligand binding. The stability of the complex and overall tertiary structural changes were characterized using root-mean-square deviation (RMSD), radius of gyration (Rg), root-mean-square fluctuation (RMSF) calculations Differences in the RMSF of apo and ligand-bound MAPK3 were most prominent in three major regions: (a) activation loop Asp184:Pro213 (b) MAPK3 insertion site Gly262:Ala291 and (c) loop region at the C-terminus Tyr334:Pro356. Lower values of RMSF for the HQ068-bound protein at the activation loop suggest that HQ068 binding stabilizes MAPK3 in a different conformation in this region compared to the apo protein. Free energy calculations of the asernestioside B-MAPK3 complex revealed key residues contributing to the interaction, which include Pro264, Gln 266, Asp268 and Thr288. These key residues may play an integral role in the binding of selective modulators or substrates of extracellular signal-regulated kinase (ERK) within the MAPK cascade. Overall, this study provides a mechanistic overview of compounds from HGWT. Modelling predicted that asernestioside B may act with high potency against MAPK3, while exhibiting a favourable ADMET profile, and this compound should be explored as a potential agent to alleviate SRNW-related symptoms in future studies.

Identification and validation of new ERK substrates by phosphoproteomic technologies including Phos-tag SDS-PAGE

The extracellular signal-regulated kinase (ERK), a member of the mitogen-activated protein (MAP) kinase family, governs various cellular processes by phosphorylating a large set of substrates. Although many studies have expanded the number of ERK substrates, it is likely that additional substrates remain to be discovered. Here we have employed a quantitative phosphoproteomic approach to explore novel ERK substrates in NIH3T3 fibroblasts stably expressing a fusion protein between B-Raf and estrogen receptor. Among ERK-dependent phosphorylation targets, we focused on NGFI-A-binding protein 2 (Nab2), forkhead box protein K1 (Foxk1), and Disks large-associated protein 5 (Dlgap5/HURP). Phos-tag SDS-PAGE followed by Western blotting confirmed ERK-dependent phosphorylation of these three proteins in cells. Phos-tag SDS-PAGE of in vitro kinase assay samples revealed high degrees of phosphorylation of these proteins by active ERK. Furthermore, in-gel digestion of the phosphorylated protein bands from Phos-tag SDS-PAGE followed by LC-MS/MS indicated that active ERK directly phosphorylates the same sites in vitro as those observed in cells. This study demonstrates the usefulness of Phos-tag SDS-PAGE for validation of candidate substrates of protein kinases. SignificanceLabel-free quantitative phosphoproteomics identified 1439 phosphopeptides derived from 840 proteins that were significantly increased by ERK activation in mouse fibroblasts. Through gene ontology and pathway analysis, we selected three proteins involved in transcriptional regulation and/or tumorigenesis. The identified phosphorylation sites of these proteins conform to the ERK consensus motif and were directly phosphorylated by active ERK in vitro. Phos-tag SDS-PAGE was useful for detecting ERK-mediated phosphorylation of these substrates both in cells and in vitro. Further characterization of these new ERK substrates will be needed to better understand the ERK signaling pathway, and our phosphoproteomic data provide useful information for studying downstream substrates of ERK.

F-box Protein βTrCP1 Is a Substrate of Extracellular Signal-regulated Kinase 2.

F-box proteins, consisting of 69 members which are organized into the three subclasses FBXW, FBXL, and FBXO, are the substrate specific recognition subunits of the SKP1-Cullin 1-F-box protein E3 ligase complex. Although βTrCP 1 and 2, members of the FBXW subfamily, are known to regulate some protein stability, molecular mechanisms by which these proteins can recognize proper substrates are unknown. In this study, it was found that βTrCP1 showed strong interaction with members of mitogen-activated protein kinases. Although extracellular signal-regulated kinase (ERK) 3, p38β, and p38δ showed weak interactions, ERK2 specifically interacted with βTrCP1 as assessed by immunoprecipitation. In interaction domain determination experiments, we found that ERK2 interacted with two independent ERK docking sites located in the F-box domain and linker domain, but not the WD40 domain, of βTrCP1. Notably, mutations of βTrCP1 at the ERK docking sites abolished the interaction with ERK2. βTrCP1 underwent phosphorylation by EGF stimulation, while the presence of the mitogen-activated protein kinase kinases inhibitor U0126, genetic silencing by sh-ERK2, and mutation of the ERK docking site of βTrCP1 inhibited phosphorylation. This inhibition of βTrCP1 phosphorylation resulted in a shortened half-life and low protein levels. These results suggest that ERK2-mediated βTrCP1 phosphorylation may induce the destabilization of βTrCP1.

Global Identification of ERK Substrates by Phosphoproteomics Based on IMAC and 2D-DIGE.

Extracellular signal-regulated kinase (ERK) regulates various cellular functions through phosphorylation of numerous downstream substrates, which have not yet been fully characterized. To date, several phosphoproteomic approaches have been employed to identify novel substrates for ERK. In this chapter, we describe a method to globally identify ERK substrates by combining immobilized metal affinity chromatography (IMAC) and two-dimensional difference gel electrophoresis (2D-DIGE) followed by mass spectrometry. Phosphoprotein enrichment by IMAC enables the subsequent detection of many protein spots with different fluorescence intensities between ERK-inhibited and -activated cells in 2D-DIGE analysis. Furthermore, the advanced sensitivity and resolution of liquid chromatography coupled with tandem mass spectrometry allow for a direct identification of proteins obtained from silver-stained 2D-DIGE gels. Validation experiments such as Phos-tag Western blotting are important steps to further elucidate the functional roles of ERK-mediated phosphorylation of these newly identified substrates.

Discovering Functional ERK Substrates Regulating Caenorhabditis elegans Germline Development.

The Rat Sarcoma (RAS) GTPAse-mediated extracellular signal-regulated kinase (ERK) pathway regulates multiple biological processes across metazoans. In particular during Caenorhabditis elegans oogenesis, ERK signaling has been shown to regulate over seven distinct biological processes in a temporal and sequential manner. To fully elucidate how ERK signaling cascade orchestrates these different biological processes in vivo, identification of the direct functional substrates of the pathway is critical. This chapter describes the methods that were used to identify ERK substrates in a global manner and study their functions in the germline. These approaches can also be generally applied to study ERK-dependent biological processes in other systems.

Kazinol-E is a specific inhibitor of ERK that suppresses the enrichment of a breast cancer stem-like cell population

Growing evidence shows that cancer stem-like cells (CSLCs) contribute to breast cancer recurrence and to its resistance to conventional therapies. The extracellular signal-regulated kinase (ERK) signaling pathway is a major determinant in the control of diverse cellular processes, including the maintenance of CSLCs. In this study, we found that Kazinol-E, an antioxidant flavan from Broussonetia kazinoki, decreased the CSLC population of a breast cancer cell line, MCF7. The CSLC population, characterized by CD44 high/CD24 low expression or by high Aldehyde dehydrogenase 1 activity, was decreased by a concentration of Kazinol-E that did not affect the growth of bulk-cultured MCF7 cells. Kazinol-E did not decrease EGF-induced ERK phosphorylation in CSLCs, but did block the phosphorylation of an ERK substrate, p90RSK2, at Thr359/Ser363. We further demonstrated that EGF-induced ERK activity was blocked by Kazinol-E in a wild-type K-Ras-expressing non-small cell lung cancer cell line H226B. An in vitro kinase assay with purified ERK1 and p90RSK2 as its substrate demonstrated a direct inhibition of ERK activity by Kazinol E. Additionally, a the molecular docking study provided putative binding modes of Kazinol-E into the ATP binding pocket of ERK1 Collectively, these results suggest that Kazinol-E is a direct inhibitor of ERK1, and more studies are warranted to develop this reagent for therapeutic breast CSLC targeting.

Nerve Growth Factor Regulates Transient Receptor Potential Vanilloid 2 via Extracellular Signal-Regulated Kinase Signaling To Enhance Neurite Outgrowth in Developing Neurons.

Neurite outgrowth is key to the formation of functional circuits during neuronal development. Neurotrophins, including nerve growth factor (NGF), increase neurite outgrowth in part by altering the function and expression of Ca(2+)-permeable cation channels. Here we report that transient receptor potential vanilloid 2 (TRPV2) is an intracellular Ca(2+)-permeable TRPV channel upregulated by NGF via the mitogen-activated protein kinase (MAPK) signaling pathway to augment neurite outgrowth. TRPV2 colocalized with Rab7, a late endosome protein, in addition to TrkA and activated extracellular signal-regulated kinase (ERK) in neurites, indicating that the channel is closely associated with signaling endosomes. In line with these results, we showed that TRPV2 acts as an ERK substrate and identified the motifs necessary for phosphorylation of TRPV2 by ERK. Furthermore, neurite length, TRPV2 expression, and TRPV2-mediated Ca(2+) signals were reduced by mutagenesis of these key ERK phosphorylation sites. Based on these findings, we identified a previously uncharacterized mechanism by which ERK controls TRPV2-mediated Ca(2+) signals in developing neurons and further establish TRPV2 as a critical intracellular ion channel in neuronal function.

Melanocortin 4 receptor activates ERK-cFos pathway to increase brain-derived neurotrophic factor expression in rat astrocytes and hypothalamus

Melanocortins are neuropeptides with well recognized anti-inflammatory and anti-apoptotic effects in the brain. Of the five melanocortin receptors (MCR), MC4R is abundantly expressed in the brain and is the only MCR present in astrocytes. We have previously shown that MC4R activation by the α-melanocyte stimulating hormone (α-MSH) analog, NDP-MSH, increased brain-derived neurotrophic factor (BDNF) expression through the classic cAMP–Protein kinase A–cAMP responsive element binding protein pathway in rat astrocytes. Now, we examined the participation of the mitogen activated protein kinases pathway in MC4R signaling. Rat cultured astrocytes treated with NDP-MSH 1 µM for 1 h showed increased BDNF expression. Inhibition of extracellular signal-regulated kinase (ERK) and ribosomal p90 S6 kinase (RSK), an ERK substrate, but not of p38 or JNK, prevented the increase in BDNF expression induced by NDP-MSH. Activation of MC4R increased cFos expression, a target of both ERK and RSK. ERK activation by MC4R involves cAMP, phosphoinositide-3 kinase (PI3K) and the non receptor tyrosine kinase, Src. Both PI3K and Src inhibition abolished NDP-MSH-induced BDNF expression. Moreover, we found that intraperitoneal injection of α-MSH induces BDNF and MC4R expression and activates ERK and cFos in male rat hypothalamus. Our results show for the first time that MC4R-induced BDNF expression in astrocytes involves ERK-RSK-cFos pathway which is dependent on PI3K and Src, and that melanocortins induce BDNF expression and ERK-cFos activation in rat hypothalamus.

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

Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which (18)O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening.

OCT1 is a determinant of synbindin-related ERK signalling with independent prognostic significance in gastric cancer

ObjectiveOctamer transcription factor 1 (OCT1) was found to be expressed in intestinal metaplasia and gastric cancer (GC), but the exact roles of OCT1 in GC remain unclear. The objective of...

ERK phosphorylation of MED14 in promoter complexes during mitogen-induced gene activation by Elk-1

The ETS domain transcription factor Elk-1 stimulates expression of immediate early genes (IEGs) in response to mitogens. These events require phosphorylation of Elk-1 by extracellular signal-regulated kinase (ERK) and phosphorylation-dependent interaction of Elk-1 with co-activators, including histone acetyltransferases and the Mediator complex. Elk-1 also recruits ERK to the promoters of its target genes, suggesting that ERK phosphorylates additional substrates in transcription complexes at mitogen-responsive promoters. Here we report that MED14, a core subunit of the Mediator, is a bona fide ERK substrate and identify serine 986 (S986) within a serine-proline rich region of MED14 as the major ERK phosphorylation site. Mitogens induced phosphorylation of MED14 on S986 at IEG promoters; RNAi knockdown of MED14 reduced CDK8 and RNA polymerase II (RNAPII) recruitment, RNAPII C-terminal domain phosphorylation and impaired activation of IEG transcription. A single alanine substitution at S986 reduced activation of an E26 (ETS)-responsive reporter by oncogenic Ras and mitogen-induced, Elk-1-dependent transcription, whereas activities of other transcriptional activators were unaffected. We also demonstrate that Elk-1 can associate with MED14 independently of MED23, which may facilitate phosphorylation of MED14 by ERK to impart a positive and selective impact on mitogen-responsive gene expression.

Epidermal Growth Factor Stimulates Extracellular-Signal Regulated Kinase Phosphorylation of a Novel Site on Cytoplasmic Dynein Intermediate Chain 2

Extracellular-signal regulated kinase (ERK) signaling is required for a multitude of physiological and patho-physiological processes. However, the identities of the proteins that ERK phosphorylates to elicit these responses are incompletely known. Using an affinity purification methodology of general utility, here we identify cytoplasmic dynein intermediate chain 2 (DYNC1I-2, IC-2) as a novel substrate for ERK following epidermal growth factor receptor stimulation of fibroblasts. IC-2 is a subunit of cytoplasmic dynein, a minus-end directed motor protein necessary for transport of diverse cargos along microtubules. Emerging data support the hypothesis that post-translational modification regulates dynein but the signaling mechanisms used are currently unknown. We find that ERK phosphorylates IC-2 on a novel, highly conserved Serine residue proximal to the binding site for the p150Glued subunit of the cargo adapter dynactin. Surprisingly, neither constitutive phosphorylation nor a phosphomimetic substitution of this Serine influences binding of p150Glued to IC-2. These data suggest that ERK phosphorylation of IC-2 regulates dynein function through mechanisms other than its interaction with dynactin.

A kinetic model of ERK cyclic pathway on substrate control

Extracellular signal-regulated kinase (ERK) is a key factor in the widely used signaling cascade of phosphorylation–dephosphorylation cycles and plays pivotal roles in many aspects of biological processes. Experimental studies in yeast and in Drosophila embryo have suggested that the phosphorylation and spatial localization of ERK are influenced by the level of its downstream substrates. However, the mechanism, through which these substrates control properties of ERK signaling, has been unclear. I propose a mass–action kinetic model of ERK cycle with its substrate, and demonstrate that the substrate can modulate the ERK activity by directly interacting with ERK. The model shows that the addition of substrate controls the level of ERK phosphorylation positively or negatively, depending on the balance between dissociation constants of ERK–substrate interaction and properties of ERK cyclic signaling in the absence of the substrate. In addition, by considering cellular compartments, cytosol and nucleus, the substrate can lead to nuclear accumulation of ERK, suggesting that the substrate can act as a nuclear anchor of ERK. The model gives a possible mechanism that can account for substrate-mediated modulation of ERK signaling.

Interactions and phosphorylation of postsynaptic density 93 (PSD-93) by extracellular signal-regulated kinase (ERK)

Postsynaptic density 93 (PSD-93) is a protein enriched at postsynaptic sites. As a key scaffolding protein, PSD-93 forms complexes with the clustering of various synaptic proteins to construct postsynaptic signaling networks and control synaptic transmission. Extracellular signal-regulated kinase (ERK) is a prototypic member of a serine/threonine protein kinase family known as mitogen-activated protein kinase (MAPK). This kinase, especially ERK2 isoform, noticeably resides in peripheral structures of neurons, such as dendritic spines and postsynaptic density areas, in addition to its distribution in the cytoplasm and nucleus, although little is known about specific substrates of ERK at synaptic sites. In this study, we found that synaptic PSD-93 is a direct target of ERK. This was demonstrated by direct protein–protein interactions between purified ERK2 and PSD-93 in vitro. The accurate ERK2-binding region seems to locate at an N-terminal region of PSD-93. In adult rat striatal neurons in vivo, native ERK from synaptosomal fractions also associated with PSD-93. In phosphorylation assays, active ERK2 phosphorylated PSD-93. An accurate phosphorylation site was identified at a serine site (S323). In striatal neurons, immunoprecipitated PSD-93 showed basal phosphorylation at an ERK-sensitive site. Our data provide evidence supporting PSD-93 as a new substrate of the synaptic species of ERK. ERK2 possesses the ability to interact with PSD-93 and phosphorylate PSD-93 at a specific site.

Role of Palladin Phosphorylation by Extracellular Signal-Regulated Kinase in Cell Migration

Phosphorylation of actin-binding proteins plays a pivotal role in the remodeling of the actin cytoskeleton to regulate cell migration. Palladin is an actin-binding protein that is phosphorylated by growth factor stimulation; however, the identity of the involved protein kinases remains elusive. In this study, we report that palladin is a novel substrate of extracellular signal-regulated kinase (ERK). Suppression of ERK activation by a chemical inhibitor reduced palladin phosphorylation, and expression of active MEK alone was sufficient for phosphorylation. In addition, an in vitro kinase assay demonstrated direct palladin phosphorylation by ERK. We found that Ser77 and Ser197 are essential residues for phosphorylation. Although the phosphorylation of these residues was not required for actin cytoskeletal organization, we found that expression of non-phosphorylated palladin enhanced cell migration. Finally, we show that phosphorylation inhibits the palladin association with Abl tyrosine kinase. Taken together, our results indicate that palladin phosphorylation by ERK has an anti-migratory function, possibly by modulating interactions with molecules that regulate cell migration.

Altered ERK/MAPK signaling in the hippocampus of the mrsk2_KO mouse model of Coffin‐Lowry syndrome

Coffin-Lowry syndrome is a syndromic form of mental retardation caused by mutations of the Rps6ka3 gene encoding ribosomal s6 kinase (RSK)2. RSK2 belongs to a family containing four members in mammals: RSK1-4. RSKs are serine/threonine kinases and cytosolic substrates of extracellular signal-regulated kinase (ERK) in the Ras/MAPK signaling pathway. RSK2 is highly expressed in the hippocampus, and mrsk2_KO mice display spatial learning and memory impairment. In the present study, we provide evidence of abnormally increased phosphorylation of ERK1/2 in the hippocampus of mrsk2_KO mice. Further studies based on cultured hippocampal neurons revealed that glutamate activates ERK1/2 and RSKs, and confirmed a stronger activation of ERK1/2 in mrsk2_KO neurons than in WT cells. We, thus, provide further evidence that RSK2 exerts a feedback inhibitory effect on the ERK1/2 pathway. We also observed a transient sequestration of P-ERK1/2 in the cytoplasm upon glutamate stimulation. In addition, the transcription factors cAMP response element binding and Ets LiKe gene1 show over-activation in RSK2-deficient neurons. Finally, c-Fos, Zif268 and Arc were significantly over-expressed in mrsk2_KO neurons upon glutamate stimulation. Importantly, the increased phosphorylation of other RSK family members observed in mutant neurons was unable to compensate for RSK2 deficiency. This aberrant ERK1/2 signaling can influence various neuronal functions, and thus play a significant role in cognitive dysfunction in mrsk2_KO mice and in the Coffin-Lowry syndrome.

A Plate-Based Assay to Measure Cellular ERK Substrate Phosphorylation: Utility for Drug Discovery of the MAPK-Signaling Cascade

The Ras, Raf, mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated kinase (ERK) signaling cascade is critically involved in cellular signaling with activating mutations in Ras and Raf present in many human tumors. Each constituent of this pathway is considered an important target for pharmaceutical intervention. The terminal kinase ERK is known to phosphorylate p90RSK among myriad substrates, yet robust plate-based high-throughput cellular assays monitoring such activity are not commercially available. In this study, we have utilized the Meso Scale Discovery platform to develop a plate-based assay to monitor the level of phosphorylation of p90RSK. This method is highly robust and can be used to evaluate a large number of inhibitors of ERK, MEK, or Raf in a variety of cellular backgrounds. Furthermore, this assay can be used to quantify the level of phospho-p90RSK in tumor lysates to function as a valuable pharmacodynamic readout.

AMP-activated Protein Kinase Antagonizes Pro-apoptotic Extracellular Signal-regulated Kinase Activation by Inducing Dual-specificity Protein Phosphatases in Response to Glucose Deprivation in HCT116 Carcinoma

Mitogen-activated protein kinase (MAPK) pathways are involved in the regulation of cellular responses, including cell proliferation, differentiation, cell growth, and apoptosis. Because these responses are tightly related to cellular energy level, AMP-activated protein kinase (AMPK), which plays an essential role in energy homeostasis, has emerged as another key regulator. In the present study, we demonstrate a novel signal network between AMPK and MAPK in HCT116 human colon carcinoma. Glucose deprivation activated AMPK and three MAPK subfamilies, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal kinase (JNK), and p38 MAPK. Under these conditions, inhibition of endogenous AMPK by expressing a dominant-negative form significantly potentiated ERK activation, indicating that glucose deprivation-induced AMPK is specifically antagonizing ERK activity in HCT116 cells. Moreover, we provide novel evidence that AMPK activity is critical for p53-dependent expression of dual-specificity phosphatase (DUSP) 1 & 2, which are negative regulators of ERK. Notably, ERK exhibits pro-apoptotic effects in HCT116 cells under glucose deprivation. Collectively, our data suggest that AMPK protects HCT116 cancer cells from glucose deprivation, in part, via inducing DUSPs, which suppresses pro-apoptotic ERK, further implying that a signal network between AMPK and ERK is a critical regulatory point in coupling the energy status of the cell to the regulation of cell survival.

Novel extracellular‐signal regulated kinase (ERK) substrate inhibitors mediate apoptotic signaling

ERK proteins promote cancer cell proliferation and survival through substrate phosphorylation events. Thus, selective inhibition of substrates involved in these mechanisms provides promising targets for anti‐cancer therapies. Small molecules designed to interfere with ERK docking domains involved in substrate interactions were identified using computer‐aided drug design (CADD). A total of 184 compounds were tested in HeLa cells. Upon treatment, 35 compounds caused the induction of apoptotic signaling as measured by poly (ADP‐ribose) polymerase (PARP) cleavage. Importantly, these effects were not observed in non‐transformed cells. To evaluate the mechanism of apoptosis induction, ERK‐mediated Caspase‐9 phosphorylation, which prevents activation of the intrinsic apoptotic pathway, was inhibited by several of the active compounds. However, other active compounds inhibited Bim phosphorylation, a pro‐apoptotic protein whose ERK‐mediated phosphorylation results in proteosomal degradation. Moreover, induction of apoptotic signaling by the active compounds did not appear to be due to off target effects on Akt kinase, which also mediates pro‐survival mechanisms. Taken together, selective inhibition of ERK substrates promote apoptosis in HeLa cells, suggesting a novel approach in the development of new anti‐cancer therapeutics. (Supported by NIH Grant # CA120215‐01 to PS & CA120215‐02S to SB)