Reprogramming of Glutamine Amino Acid Transporters Expression and Prognostic Significance in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is the most prevalent primary liver malignancy and is a major cause of cancer-related mortality in the world. This study aimed to characterize glutamine amino acid transporter expression profiles in HCC compared to those of normal liver cells. In vitro and in vivo models of HCC were studied using qPCR, whereas the prognostic significance of glutamine transporter expression levels within patient tumors was analyzed through RNAseq. Solute carrier (SLC) 1A5 and SLC38A2 were targeted through siRNA or gamma-p-nitroanilide (GPNA). HCC cells depended on exogenous glutamine for optimal survival and growth. Murine HCC cells showed superior glutamine uptake rate than normal hepatocytes (p < 0.0001). HCC manifested a global reprogramming of glutamine transporters compared to normal liver: SLC38A3 levels decreased, whereas SLC38A1, SLC7A6, and SLC1A5 levels increased. Also, decreased SLC6A14 and SLC38A3 levels or increased SLC38A1, SLC7A6, and SLC1A5 levels predicted worse survival outcomes (all p < 0.05). Knockdown of SLC1A5 and/or SLC38A2 expression in human Huh7 and Hep3B HCC cells, as well as GPNA-mediated inhibition, significantly decreased the uptake of glutamine; combined SLC1A5 and SLC38A2 targeting had the most considerable impact (all p < 0.05). This study revealed glutamine transporter reprogramming as a novel hallmark of HCC and that such expression profiles are clinically significant.

Phosphoproteome dynamics reveal novel ERK1/2 MAP kinase substrates with broad spectrum of functions.

The ERK1/2 MAP kinase pathway is an evolutionarily conserved signaling module that controls many fundamental physiological processes. Deregulated activity of ERK1/2 MAP kinases is associated with developmental syndromes and several human diseases. Despite the importance of this pathway, a comprehensive picture of the natural substrate repertoire and biochemical mechanisms regulated by ERK1/2 is still lacking. In this study, we used large-scale quantitative phosphoproteomics and bioinformatics analyses to identify novel candidate ERK1/2 substrates based on their phosphorylation signature and kinetic profiles in epithelial cells. We identified a total of 7936 phosphorylation sites within 1861 proteins, of which 155 classify as candidate ERK1/2 substrates, including 128 new targets. Candidate ERK1/2 substrates are involved in diverse cellular processes including transcriptional regulation, chromatin remodeling, RNA splicing, cytoskeleton dynamics, cellular junctions and cell signaling. Detailed characterization of one newly identified substrate, the transcriptional regulator JunB, revealed that ERK1/2 phosphorylate JunB on a serine adjacent to the DNA-binding domain, resulting in increased DNA-binding affinity and transcriptional activity. Our study expands the spectrum of cellular functions controlled by ERK1/2 kinases.

Syngeneic mouse model of YES-driven metastatic and proliferative hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is a disease of high unmet medical need that has become a global health problem. The development of targeted therapies for HCC has been hindered by the incomplete understanding of HCC pathogenesis and the limited number of relevant preclinical animal models. We recently unveiled a previously uncharacterized YES kinase (encoded by YES1)-dependent oncogenic signaling pathway in HCC. To model this subset of HCC, we established a series of syngeneic cell lines from liver tumors of transgenic mice expressing activated human YES. The resulting cell lines (referred to as HepYF) were enriched for expression of stem cell and progenitor markers, proliferated rapidly, and were characterized by high SRC family kinase (SFK) activity and activated mitogenic signaling pathways. Transcriptomic analysis indicated that HepYF cells are representative of the most aggressive proliferation class G3 subgroup of HCC. HepYF cells formed rapidly growing metastatic tumors upon orthotopic implantation into syngeneic hosts. Treatment with sorafenib or the SFK inhibitor dasatinib markedly inhibited the growth of HepYF tumors. The new HepYF HCC cell lines provide relevant preclinical models to study the pathogenesis of HCC and test novel small-molecule inhibitor and immunotherapy approaches.

Discovery of Benzodiazepine-Based Inhibitors of the E2 Enzyme UBCH10 from a Cell-Based p21 Degradation Screen.

p21Cip1 (p21) is a universal cyclin-dependent kinase (CDK) inhibitor that halts cell proliferation and tumor growth by multiple mechanisms. The expression of p21 is often downregulated in cancer cells as a result of the loss of function of transcriptional activators, such as p53, or the increased degradation rate of the protein. To identify small molecules that block the ubiquitin-mediated degradation of p21 as a future avenue for cancer drug discovery, we have screened a compound library using a cell-based reporter assay of p21 degradation. This led to the identification of a benzodiazepine series of molecules that induce the accumulation of p21 in cells. Using a chemical proteomic strategy, we identified the ubiquitin-conjugating enzyme UBCH10 as a cellular target of this benzodiazepine series. We show that an optimized benzodiazepine analogue inhibits UBCH10 ubiquitin-conjugating activity and substrate proteolysis by the anaphase-promoting complex.

Development of a high-throughput assay to identify inhibitors of the ubiquitin-conjugating enzyme UBCH10.

UBCH10 is an ubiquitin-conjugating enzyme (E2) of the anaphase-promoting complex E3 ligase, a key regulator of the cell cycle. The UBCH10 gene and protein are frequently upregulated in multiple solid tumors, associated with an unfavorable outcome. Accumulating evidence from studies of human cancer cell lines, mouse transgenic models, and analyses of clinical samples suggest that UBCH10 is a potential cancer drug target. No small molecule inhibitor of UBCH10 has been reported in the literature. Here, we described the development and optimization of a novel time-resolved fluorescence resonance energy transfer (TR-FRET) UBCH10 assay based on the self-polyubiquitination of the enzyme in the absence of E3. The homogenous assay is robust, sensitive, and scalable to different multi-well formats for high-throughput screening (HTS). We demonstrate the suitability of the TR-FRET assay to identify chemical inhibitors of UBCH10 in a pilot HTS campaign.

Signaling by the tyrosine kinase Yes promotes liver cancer development.

Most patients with hepatocellular carcinoma (HCC) are diagnosed at a late stage and have few therapeutic options and a poor prognosis. This is due to the lack of clearly defined underlying mechanisms or a dominant oncogene that can be targeted pharmacologically, unlike in other cancer types. Here, we report the identification of a previously uncharacterized oncogenic signaling pathway in HCC that is mediated by the tyrosine kinase Yes. Using genetic and pharmacological interventions in cellular and mouse models of HCC, we showed that Yes activity was necessary for HCC cell proliferation. Transgenic expression of activated Yes in mouse hepatocytes was sufficient to induce liver tumorigenesis. Yes phosphorylated the transcriptional coactivators YAP and TAZ (YAP/TAZ), promoting their nuclear accumulation and transcriptional activity in HCC cells and liver tumors. We also showed that YAP/TAZ were effectors of the Yes-dependent oncogenic transformation of hepatocytes. Src family kinase activation correlated with the tyrosine phosphorylation and nuclear localization of YAP in human HCC and was associated with increased tumor burden in mice. Specifically, high Yes activity predicted shorter overall survival in patients with HCC. Thus, our findings identify Yes as a potential therapeutic target in HCC.

ProteoConnections: a bioinformatics platform to facilitate proteome and phosphoproteome analyses.

Novel and improved computational tools are required to transform large-scale proteomics data into valuable information of biological relevance. To this end, we developed ProteoConnections, a bioinformatics platform tailored to address the pressing needs of proteomics analyses. The primary focus of this platform is to organize peptide and protein identifications, evaluate the quality of the acquired data set, profile abundance changes, and accelerate data interpretation. Peptide and protein identifications are stored into a relational database to facilitate data mining and to evaluate the quality of data sets using graphical reports. We integrated databases of known PTMs and other bioinformatics tools to facilitate the analysis of phosphoproteomics data sets and to provide insights for subsequent biological validation experiments. Phosphorylation sites are also annotated according to kinase consensus motifs, contextual environment, protein domains, binding motifs, and evolutionary conservation across different species. The practical application of ProteoConnections is further demonstrated for the analysis of the phosphoproteomics data sets from rat intestinal IEC-6 cells where we identified 9615 phosphorylation sites on 2108 phosphoproteins. Combined proteomics and bioinformatics analyses revealed valuable biological insights on the regulation of phosphoprotein functions via the introduction of new binding sites on scaffold proteins or the modulation of protein-protein, protein-DNA, or protein-RNA interactions. Quantitative proteomics data can be integrated into ProteoConnections to determine the changes in protein phosphorylation under different cell stimulation conditions or kinase inhibitors, as demonstrated here for the MEK inhibitor PD184352.

Effects of angiotensin-converting enzyme inhibitor versus valsartan on cellular signaling events in heart transplant.

BACKGROUND: Angiotensin-converting enzyme (ACE) inhibitors and angiotensin receptor blockers (ARBs) provide similar biologic effects in model systems and similar clinical impacts in humans. The changes in the cardiac angiotensin system signaling pathways in the human heart in response to ACE inhibitors versus ARBs have been incompletely studied. OBJECTIVE: To investigate the effects of ACE inhibitors versus valsartan on the angiotensin II signal transduction pathways in the transplanted human heart. METHODS: Twenty-seven stable cardiac transplant recipients were randomized to remain on ACE inhibitor therapy (n = 8) or to receive valsartan (n = 19). Two additional endomyocardial biopsy samples were obtained at baseline and after 9 months of therapy. The expression of cardiac angiotensin type I and II receptors and atrial natriuretic factor (ANF) was measured by quantitative polymerase chain reaction. The expression and phosphorylation levels of selected signal transduction pathways were analyzed by immunoblotting. RESULTS: The mean dose of valsartan was 114 +/- 41 mg/day. The use of valsartan resulted in a similar impact on blood pressure and biochemistry profile. There were no significant changes in the expression of angiotensin type I and II receptors and ANF with valsartan. Similarly, no significant changes in the expression and phosphorylation of Jun N-terminal kinase, extracellular signal-regulated kinase 1 and 2, and p38 mitogen-activated protein kinases or AKT, and mammalian target of rapamycin was observed in the valsartan-treated group. CONCLUSIONS: Valsartan use is associated with similar clinical and molecular cardiac effects as ACE inhibitor therapy in stable long-term cardiac transplant recipients.

Activation of MEK1 or MEK2 isoform is sufficient to fully transform intestinal epithelial cells and induce the formation of metastatic tumors.

BACKGROUND: The Ras-dependent ERK1/2 MAP kinase signaling pathway plays a central role in cell proliferation control and is frequently activated in human colorectal cancer. Small-molecule inhibitors of MEK1/MEK2 are therefore viewed as attractive drug candidates for the targeted therapy of this malignancy. However, the exact contribution of MEK1 and MEK2 to the pathogenesis of colorectal cancer remains to be established. METHODS: Wild type and constitutively active forms of MEK1 and MEK2 were ectopically expressed by retroviral gene transfer in the normal intestinal epithelial cell line IEC-6. We studied the impact of MEK1 and MEK2 activation on cellular morphology, cell proliferation, survival, migration, invasiveness, and tumorigenesis in mice. RNA interference was used to test the requirement for MEK1 and MEK2 function in maintaining the proliferation of human colorectal cancer cells. RESULTS: We found that expression of activated MEK1 or MEK2 is sufficient to morphologically transform intestinal epithelial cells, dysregulate cell proliferation and induce the formation of high-grade adenocarcinomas after orthotopic transplantation in mice. A large proportion of these intestinal tumors metastasize to the liver and lung. Mechanistically, activation of MEK1 or MEK2 up-regulates the expression of matrix metalloproteinases, promotes invasiveness and protects cells from undergoing anoikis. Importantly, we show that silencing of MEK2 expression completely suppresses the proliferation of human colon carcinoma cell lines, whereas inactivation of MEK1 has a much weaker effect. CONCLUSION: MEK1 and MEK2 isoforms have similar transforming properties and are able to induce the formation of metastatic intestinal tumors in mice. Our results suggest that MEK2 plays a more important role than MEK1 in sustaining the proliferation of human colorectal cancer cells.

Deubiquitinating Enzyme USP20 Regulates Extracellular Signal-Regulated Kinase 3 Stability and Biological Activity.

Extracellular signal-regulated kinase 3 (ERK3) is an atypical mitogen-activated protein kinase (MAPK) whose regulatory mechanisms and biological functions remain superficially understood. Contrary to most protein kinases, ERK3 is a highly unstable protein that is subject to dynamic regulation by the ubiquitin-proteasome system. However, the effectors that control ERK3 ubiquitination and degradation are unknown. In this study, we carried out an unbiased functional loss-of-function screen of the human deubiquitinating enzyme (DUB) family and identified ubiquitin-specific protease 20 (USP20) as a novel ERK3 regulator. USP20 interacts with and deubiquitinates ERK3 both in vitro and in intact cells. The overexpression of USP20 results in the stabilization and accumulation of the ERK3 protein, whereas USP20 depletion reduces the levels of ERK3. We found that the expression levels of ERK3 correlate with those of USP20 in various cellular contexts. Importantly, we show that USP20 regulates actin cytoskeleton organization and cell migration in a manner dependent on ERK3 expression. Our results identify USP20 as a bona fide regulator of ERK3 stability and physiological functions.

Loss of Extracellular Signal-Regulated Kinase 1/2 in the Retinal Pigment Epithelium Leads to RPE65 Decrease and Retinal Degeneration.

Recent work suggested that the activity of extracellular signal-regulated kinase 1/2 (ERK1/2) is increased in the retinal pigment epithelium (RPE) of age-related macular degeneration (ARMD) patients and therefore could be an attractive therapeutic target. Notably, ERK1/2 pathway inhibitors are used in cancer therapy, with severe and noncharacterized ocular side effects. To decipher the role of ERK1/2 in RPE cells, we conditionally disrupted the Erk1 and Erk2 genes in mouse RPE. The loss of ERK1/2 activity resulted in a significant decrease in the level of RPE65 expression, a decrease in ocular retinoid levels concomitant with low visual function, and a rapid disorganization of RPE cells, ultimately leading to retinal degeneration. Our results identify the ERK1/2 pathway as a direct regulator of the visual cycle and a critical component of the viability of RPE and photoreceptor cells. Moreover, our results caution about the need for a very fine adjustment of kinase inhibition in cancer or ARMD treatment in order to avoid ocular side effects.

Deregulated ERK1/2 MAP kinase signaling promotes aneuploidy by a Fbxw7ß-Aurora A pathway.

Aneuploidy is a common feature of human solid tumors and is often associated with poor prognosis. There is growing evidence that oncogenic signaling pathways, which are universally dysregulated in cancer, contribute to the promotion of aneuploidy. However, the mechanisms connecting signaling pathways to the execution of mitosis and cytokinesis are not well understood. Here, we show that hyperactivation of the ERK1/2 MAP kinase pathway in epithelial cells impairs cytokinesis, leading to polyploidization and aneuploidy. Mechanistically, deregulated ERK1/2 signaling specifically downregulates expression of the F-box protein Fbxw7ß, a substrate-binding subunit of the SCF(Fbxw7) ubiquitin ligase, resulting in the accumulation of the mitotic kinase Aurora A. Reduction of Aurora A levels by RNA interference or pharmacological inhibition of MEK1/2 reverts the defect in cytokinesis and decreases the frequency of abnormal cell divisions induced by oncogenic H-Ras(V12). Reciprocally, overexpression of Aurora A or silencing of Fbxw7ß phenocopies the effect of H-Ras(V12) on cell division. In vivo, conditional activation of MEK2 in the mouse intestine lowers Fbxw7ß expression, resulting in the accumulation of cells with enlarged nuclei. We propose that the ERK1/2/ Fbxw7ß/Aurora A axis identified in this study contributes to genomic instability and tumor progression.

The Rb-family protein p107 inhibits translation by a PDK1-dependent mechanism.

The Rb family of proteins, which consists of Rb, p107 and p130, are critical regulators of cell proliferation. In addition to their inhibitory effects on cell cycle progression, Rb-family proteins repress transcription by RNA polymerases I and III, and may therefore restrain cell growth. However, it is not known if Rb, p107 or p130 have direct effects on protein synthesis. Here we report that ectopic expression of p107 in rat fibroblasts markedly attenuates the stimulation of mRNA translation and global protein synthesis by serum growth factors. This effect is associated with a reduction in the phosphorylation and activation of the serine-threonine kinases Akt1 and p70 S6 kinase (S6K1), two downstream targets of phosphoinositide-dependent kinase 1 (PDK1). We show that overexpression of p107 interferes with the recruitment of PDK1 to the plasma membrane in response to growth factors. Overexpression of PDK1 restores the defect in translation elicited by p107. These results suggest that p107 restricts cell growth by interfering with the phosphoinositide 3-kinase (PI3K) signaling pathway.

MEK1-ERK2 signaling pathway protects myocardium from ischemic injury in vivo.

BACKGROUND: Myocardial infarction causes a rapid and largely irreversible loss of cardiac myocytes that can lead to sudden death, ventricular dilation, and heart failure. Members of the mitogen-activated protein kinase (MAPK) signaling cascade have been implicated as important effectors of cardiac myocyte cell death in response to diverse stimuli, including ischemia-reperfusion injury. Specifically, activation of the extracellular signal-regulated kinases 1/2 (ERK1/2) has been associated with cardioprotection, likely through antagonism of apoptotic regulatory pathways. METHODS AND RESULTS: To establish a causal relationship between ERK1/2 signaling and cardioprotection, we analyzed Erk1 nullizygous gene-targeted mice, Erk2 heterozygous gene-targeted mice, and transgenic mice with activated MEK1-ERK1/2 signaling in the heart. Although MEK1 transgenic mice were largely resistant to ischemia-reperfusion injury, Erk2+/- gene-targeted mice showed enhanced infarction areas, DNA laddering, and terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling (TUNEL) compared with littermate controls. In contrast, enhanced MEK1-ERK1/2 signaling protected hearts from DNA laddering, TUNEL, and preserved hemodynamic function assessed by pressure-volume loop recordings after ischemia-reperfusion injury. CONCLUSIONS: These data are the first to demonstrate that ERK2 signaling is required to protect the myocardium from ischemia-reperfusion injury in vivo.

Erk2 signaling and early embryo stem cell self-renewal.

Differentiation of the mammalian blastocyst generates two distinct cell lineages: the trophectoderm, which contributes to the trophoblast layers of the placenta, and the inner cell mass, which forms the embryo. We and others recently demonstrated that the MAP kinase ERK2 is essential for trophoblast development. Erk2 mutant embryos fail to form extra-embryonic ectoderm and the ectoplacental cone, suggesting a role for ERK2 activation in the proliferation of trophoblast stem (TS) cells. Previous studies have documented that ERK1/2 activity is dispensable for proliferation of embryonic stem (ES) cells and rather interferes with self-renewal. Thus, signaling by the ERK1/2 MAP kinase pathway appears to be critical for the regulation of self-renewal and propagation of early embryo stem cell populations.

Towards the development of chromone-based MEK1/2 modulators.

Inhibition or allosteric modulation of mitogen-activated protein kinase kinases MEK1 and MEK2 (MEK1/2) represent a promising strategy for the discovery of new specific anticancer agents. In this paper, structure-based design, beginning from the lead compound PD98059, was used to study potential structural modifications on the chromone structure in order to obtain highly potent derivatives that target the allosteric pocket in MEK1. Subsequently, a small series of PD98059 analogs were synthesized to provide a first generation of chromone-based derivatives that inhibit the activation of MEK1 with IC50 values as low as 30 nM in vitro. Complementary cellular studies also showed that two of the compounds in the series inhibit the activity of MEK1/2 with IC50 values in the nanomolar range (73-97 nM). In addition, compounds in this series were found to inhibit the proliferation of a small panel of human cancer cell lines.

Genetic demonstration of a redundant role of extracellular signal-regulated kinase 1 (ERK1) and ERK2 mitogen-activated protein kinases in promoting fibroblast proliferation.

The extracellular signal-regulated kinase 1 and 2 (ERK1/2) mitogen-activated protein (MAP) kinase signaling pathway plays an important role in the proliferative response of mammalian cells to mitogens. However, the individual contribution of the isoforms ERK1 and ERK2 to cell proliferation control is unclear. The two ERK isoforms have similar biochemical properties and recognize the same primary sequence determinants on substrates. On the other hand, analysis of mice lacking individual ERK genes suggests that ERK1 and ERK2 may have evolved unique functions. In this study, we used a robust genetic approach to analyze the individual functions of ERK1 and ERK2 in cell proliferation using genetically matched primary embryonic fibroblasts. We show that individual loss of either ERK1 or ERK2 slows down the proliferation rate of fibroblasts to an extent reflecting the expression level of the kinase. Moreover, RNA interference-mediated silencing of ERK1 or ERK2 expression in cells genetically disrupted for the other isoform similarly reduces cell proliferation. We generated fibroblasts genetically deficient in both Erk1 and Erk2. Combined loss of ERK1 and ERK2 resulted in a complete arrest of cell proliferation associated with G(1) arrest and premature replicative senescence. Together, our findings provide compelling genetic evidence for a redundant role of ERK1 and ERK2 in promoting cell proliferation.

EGF receptor transactivation is obligatory for protein synthesis stimulation by G protein-coupled receptors.

The epidermal growth factor receptor (EGFR) was recently identified as a signal transducer of G protein-coupled receptors (GPCRs). In this study, we have examined the contribution of EGFR transactivation to the growth-promoting effect of GPCRs on vascular smooth muscle cells. Activation of the G(q)-coupled ANG II receptor or G(i)-coupled lysophosphatidic acid receptor resulted in increased tyrosine phosphorylation and activation of EGFR. Specific inhibition of EGFR kinase activity by tyrphostin AG-1478 or expression of a dominant-negative EGFR mutant abolished this response. Importantly, inhibition of EGFR function strongly attenuated the global stimulation of protein synthesis by GPCR agonists in vitro in cultured aortic smooth muscle cells and in vivo in the rat aorta and in small resistance arteries. The growth inhibition was associated with a marked reduction of extracellular signal-regulated kinase and phosphoinositide 3-kinase pathway activity and the resulting suppression of eukaryotic translation initiation factor 4E and 4E binding protein 1 phosphorylation. Our results demonstrate that EGFR transactivation is a physiologically relevant action of GPCRs linked to translational control and protein synthesis.

Torbafylline (HWA 448) inhibits enhanced skeletal muscle ubiquitin-proteasome-dependent proteolysis in cancer and septic rats.

The development of new pharmacological approaches for preventing muscle wasting in cancer is an important goal because cachectic patients display a reduced response to chemotherapy and radiotherapy. Xanthine derivatives such as pentoxifylline inhibit tumour necrosis factor-alpha (TNF) production, which has been implicated in the signalling of muscle wasting. However, the effect of pentoxifylline has been inconclusive in clinical trials. We report here the first direct evidence that daily injections of torbafylline (also known as HWA 448), another xanthine derivative, had no effect by itself on muscle proteolysis in control healthy rats. In cancer rats, the drug blocked the lipopolysaccharide-induced hyperproduction of TNF and prevented muscle wasting. In these animals HWA 448 suppressed the enhanced proteasome-dependent proteolysis, which is sensitive to the proteasome inhibitor MG132, and the accumulation of high-molecular-mass ubiquitin (Ub) conjugates in the myofibrillar fraction. The drug also normalized the enhanced muscle expression of Ub, which prevails in the atrophying muscles from cancer rats. In contrast, HWA 448 did not reduce the increased expression of either the 14 kDa Ub conjugating enzyme E2 or the ATPase and non-ATPase subunits of the 19 S regulatory complex of the 26 S proteasome, including the non-ATPase subunit S5a, which recognizes polyUb degradation signals. Finally, the drug also prevented muscle wasting in septic rats (which exhibit increased TNF production), and was much more potent than pentoxifylline or other xanthine derivatives. Taken together, the data indicate that HWA 448 is a powerful inhibitor of muscle wasting that blocks enhanced Ub-proteasome-dependent proteolysis in situations where TNF production rises, including cancer and sepsis.

An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development.

The closely related mitogen-activated protein kinase isoforms extracellular signal-regulated kinase 1 (ERK1) and ERK2 have been implicated in the control of cell proliferation, differentiation and survival. However, the specific in vivo functions of the two ERK isoforms remain to be analysed. Here, we show that disruption of the Erk2 locus leads to embryonic lethality early in mouse development after the implantation stage. Erk2 mutant embryos fail to form the ectoplacental cone and extra-embryonic ectoderm, which give rise to mature trophoblast derivatives in the fetus. Analysis of chimeric embryos showed that Erk2 functions in a cell-autonomous manner during the development of extra-embryonic cell lineages. We also found that both Erk2 and Erk1 are widely expressed throughout early-stage embryos. The inability of Erk1 to compensate for Erk2 function suggests a specific function for Erk2 in normal trophoblast development in the mouse, probably in regulating the proliferation of polar trophectoderm cells.