Characterization of the activity of human MAP kinase-interacting kinase Mnk1b

Human mitogen-activated protein (MAP) kinase interacting kinase 1b (Mnk1b) is a splice variant of human Mnk1a, which has been identified in our laboratory [A. O'Loghlen, V.M. Gonzalez, D. Pineiro, M.I. Perez-Morgado, M. Salinas, M.E. Martin, Identification and molecular characterization of Mnk1b, a splice variant of human MAP kinase-interacting kinase Mnk1, Exp. Cell Res. 299 (2004) 343-355]. Mnk1b has much higher basal eIF4E kinase activity than Mnk1a. Because Mnk1b presents different features in its C-terminus with respect to Mnk1a, we have studied in this paper the potential role of these structural differences in determining the higher basal eIF4E kinase activity as well as the subcellular localization of Mnk1b. In this paper, we demonstrate that phosphorylation of the Thr209 and Thr214 in the activation loop of Mnk1b is necessary for its activation. However, the different kinase activity between Mnk1a and Mnk1b is independent of the phosphorylation status of the activation loop residues. By deletion of the C-terminal tail in Mnk1a, we confirmed that the absence of this sequence is not responsible for the higher eIF4E kinase activity present in Mnk1b. Moreover, our findings support a crucial role of the 12 amino acids, particularly the Ala344, in the C-terminal specific region of Mnk1b (Mnk1bSR), on the kinase activity of the protein.     

Identification and characterization of a novel human APH-1b splice variant lacking exon 4

APH-1 is one of the four essential components of the presenilin-gamma-secretase complex and has two human homologs, APH-1a, and APH-1b, both of which are seven-pass membrane proteins. Here, we identified a novel splice variant of human APH-1b. This variant lacks exon 4, which encodes the entire fourth transmembrane domain. The mRNA expression of this variant was detected in most tissues at low levels. In transiently transfected cells, protein expression of the APH-1b variant was much lower than that of the wild-type. Furthermore, exogenous expression of the APH-1-interacting protein, nicastrin, significantly increased the variant protein levels. These data suggest that the APH-1b variant protein is destabilized, and implies that the fourth transmembrane domain plays an important role in the protein stability and function of APH-1.     

Identification of Dok-4b, a Dok-4 splice variant with enhanced inhibitory properties

Dok adapter proteins have been primarily implicated in negative regulation of tyrosine kinase signaling, but Dok-4 has been reported to exert both inhibitory and stimulatory effects. We have identified a splice variant of Dok-4, Dok-4b, which contains a 39 aa insert within the its C-terminal region. The approximately 45kDa Dok-4b protein was detected in several human epithelial cell lines. Based on genomic sequences, Dok-4b was also predicted to exist in primates and possibly bovines, but not in rodents or other species. Compared to Dok-4, Dok-4b inhibited the tyrosine kinase-induced activation of both Erk and Elk-1 more strongly. Truncation of the C-terminal region of Dok-4 (Dok-4 DeltaCT) also enhanced the inhibitory activity of Dok-4, whereas expression of the isolated C-terminal domain enhanced Elk-1 activation, suggesting that the N-terminus and C-terminus of Dok-4 possess opposing inhibitory and stimulatory properties, respectively, the balance of which is altered by alternative splicing of Dok-4 to Dok-b.     

Identification of additional MAP kinases activated upon PAMP treatment

Mitogen-activated protein (MAP) kinase cascades play important roles in plant immunity. Upon pathogen associated molecular pattern (PAMP) treatment, MPK3, MPK6 and MPK4 are quickly activated by upstream MKKs through phosphorylation. Western blot analysis using α-phospho-p44/42-ERK antibody suggests that additional MPKs with similar size as MPK4 are also activated upon PAMP perception. To identify these MAP kinases, 7 candidate MPKs with similar sizes as MPK4 were selected for further analysis. Transgenic plants expressing these MPKs with a ZZ-3xFLAG double tag of 17 kD were generated and analyzed by western blot. MPK1, MPK11 and MPK13 were found to be phosphorylated upon treatment with flg22. Our study revealed additional MAPKs being activated during PAMP-triggered immunity.     

Hyperglycemia-induced O-GlcNAcylation and truncation of 4E-BP1 protein in liver of a mouse model of type 1 diabetes

4E-BP1 is a protein that, in its hypophosphorylated state, binds the mRNA cap-binding protein eIF4E and represses cap-dependent mRNA translation. By doing so, it plays a major role in the regulation of gene expression by controlling the overall rate of mRNA translation as well as the selection of mRNAs for translation. Phosphorylation of 4E-BP1 causes it to release eIF4E to function in mRNA translation. 4E-BP1 is also subject to covalent addition of N-acetylglucosamine to Ser or Thr residues (O-GlcNAcylation) as well as to truncation. In the truncated form, it is both resistant to phosphorylation and able to bind eIF4E with high affinity. In the present study, Ins2(Akita/+) diabetic mice were used to test the hypothesis that hyperglycemia and elevated flux of glucose through the hexosamine biosynthetic pathway lead to increased O-GlcNAcylation and truncation of 4E-BP1 and consequently decreased eIF4E function in the liver. The amounts of both full-length and truncated 4E-BP1 bound to eIF4E were significantly elevated in the liver of diabetic as compared with non-diabetic mice. In addition, O-GlcNAcylation of both the full-length and truncated proteins was elevated by 2.5- and 5-fold, respectively. Phlorizin treatment of diabetic mice lowered blood glucose concentrations and reduced the expression and O-GlcNAcylation of 4E-BP1. Additionally, when livers were perfused in the absence of insulin, 4E-BP1 phosphorylation in the livers of diabetic mice was normalized to the control value, yet O-GlcNAcylation and the association of 4E-BP1 with eIF4E remained elevated in the liver of diabetic mice. These findings provide insight into the pathogenesis of metabolic abnormalities associated with diabetes.     

The N and C termini of the splice variants of the human mitogen-activated protein kinase-interacting kinase Mnk2 determine activity and localization

The cap-binding eukaryotic initiation factor eIF4E is phosphorylated by the mitogen-activated protein (MAP) kinase-interacting kinases (Mnk's). Three forms of the Mnk's exist in human cells: Mnk1, Mnk2a, and Mnk2b. These last two are derived from the same gene by alternative splicing and differ only at their C termini. While Mnk2a contains a MAP kinase-binding site in this region, Mnk2b lacks such a sequence and is much less readily activated by MAP kinases in vitro. Expression of Mnk2b in mammalian cells leads to increased phosphorylation of eIF4E, showing that it acts as an eIF4E kinase in vivo. While Mnk2a is cytoplasmic, a substantial amount of Mnk2b is found in the nucleus. Both enzymes contain a stretch of basic residues in their N termini that plays a role in binding to eIF4G and functions as a nuclear localization signal. Binding of eIF4G or nuclear import appears to be regulated by the C terminus of Mnk2a. Furthermore, the MAP kinase-binding site of Mnk2a regulates nuclear entry. Within the nucleus, Mnk2b and certain variants of Mnk2a that are present in the nucleus colocalize with the promyelocytic leukemia protein PML, which also binds to eIF4E.     

Identification and characterisation of a novel splice variant of the human CB1 receptor

Cannabinoid ligands are implicated in many physiological processes and to date two receptors have been identified. However, a growing body of evidence exists that suggests the presence of additional receptors. Whilst cloning the previously described hCB1a, we have identified a novel variant that we call hCB1b. Characterising these two splice variants demonstrates that they have a unique pharmacological profile and that their RNA's are expressed at low levels in a variety of tissues.     

4E-BP1 degradation and eIF4E truncation occur spatially distinctly in the porcine uterine epithelia and are features of noninvasive implantation in the pig

The implantation of the blastocyst into the endometrium is an indispensable premise for successful embryonic development. This process is regulated by maternal and embryonic signals that influence gene expression at the translational level, among other processes. Recently, we have shown that proteolytical cleavage of the prototypical 25‐kDa, mRNA cap‐binding protein eIF4E produces a stable variant with a molecular mass of approximately 23 kDa exclusively in the porcine endometrium during implantation. This is accompanied by dephosphorylation and reduction of the abundant repressor 4E‐BP1. Here, we investigate the distribution of the truncated eIF4E and of 4E‐BP1 in the porcine uterine tissue, their binding in native samples, and we analyzed eIF4E‐, eIF4G‐, and 4E‐BP1‐specific proteolytic activities. Our results show that in pigs, the truncated eIF4E is located in the endometrial luminal epithelium during implantation. Neither glandulary tissue nor stroma expressed any truncated eIF4E. The reduced abundance of 4E‐BP1 during implantation is mainly the result of decay in the glandular epithelia. Moreover, steroid replacements, in vitro protease assays, and cell lysate fractionation showed that eIF4E cleavage and 4E‐BP1 decay both depended on the ovarian steroid hormones estradiol and progestrone, but these effects are the result of different proteolytic activities. Although eIF4G cleavage also depends on calcium, stimulation by these steroids could not be established. We propose that the translation initiation process in the endometrium is differently regulated by the truncated eIF4E, utilizing different abundances of 4E‐BP1 and binding dynamic of eIF4E/4E‐BP1 in distinct forms of implantation. Mol. Reprod. Dev. 78:895–905, 2011. © 2011 Wiley Periodicals, Inc.     

Identification and characterization of a novel MAP kinase kinase kinase, MLTK

The MAPK cascades regulate a wide variety of cellular functions, including cell proliferation, differentiation, and stress responses. Here we have identified a novel MAP kinase kinase kinase (MAPKKK), termed MLTK (for MLK-like mitogen-activated protein triple kinase), whose expression is increased by activation of the ERK/MAPK pathway. There are two alternatively spliced forms of MLTK, MLTKalpha and MLTKbeta. When overexpressed in cells, both MLTKalpha and MLTKbeta are able to activate the ERK, JNK/SAPK, p38, and ERK5 pathways. Moreover, both MLTKalpha and MLTKbeta are activated in response to osmotic shock with hyperosmolar media through autophosphorylation. Remarkably, expression of MLTKalpha, but not MLTKbeta, in Swiss 3T3 cells results in the disruption of actin stress fibers and dramatic morphological changes. A kinase-dead form of MLTKalpha does not cause these phenomena. Inhibition of the p38 pathway significantly blocks MLTKalpha-induced stress fiber disruption and morphological changes. These results suggest that MLTK is a stress-activated MAPKKK that may be involved in the regulation of actin organization.     

The synergistic inhibition of breast cancer proliferation by combined treatment with 4EGI-1 and MK2206

Cap-dependent translation is a potential cancer-related target (oncotarget) due to its critical role in cancer initiation and progression. 4EGI-1, an inhibitor of eIF4E/eIF4G interaction, was discovered by screening chemical libraries of small molecules. 4EGI-1 inhibits cap-dependent translation initiation by impairing the assembly of the eIF4E/eIF4G complex, and therefore is a potential anti-cancer agent. Here, we report that 4EGI-1 also inhibits mTORC1 signaling independent of its inhibitory role on cap-dependent translation initiation. The inhibition of mTORC1 signaling by 4EGI-1 activates Akt due to both abrogation of the negative feedback loops from mTORC1 to PI3K and activation of mTORC2. We further validated that mTORC2 activity is required for 4EGI-1-mediated Akt activation. The activated Akt counteracted the anticancer effects of 4EGI-1. In support of this model, inhibition of Akt potentiates the antitumor activity of 4EGI-1 both in vitro and in a xenograft mouse model in vivo. Our results suggest that a combination of 4EGI-1and Akt inhibitor is a rational approach for the treatment of cancer.     

The synergistic inhibition of breast cancer proliferation by combined treatment with 4EGI-1 and MK2206

Cap-dependent translation is a potential cancer-related target (oncotarget) due to its critical role in cancer initiation and progression. 4EGI-1, an inhibitor of eIF4E/eIF4G interaction, was discovered by screening chemical libraries of small molecules. 4EGI-1 inhibits cap-dependent translation initiation by impairing the assembly of the eIF4E/eIF4G complex, and therefore is a potential anti-cancer agent. Here, we report that 4EGI-1 also inhibits mTORC1 signaling independent of its inhibitory role on cap-dependent translation initiation. The inhibition of mTORC1 signaling by 4EGI-1 activates Akt due to both abrogation of the negative feedback loops from mTORC1 to PI3K and activation of mTORC2. We further validated that mTORC2 activity is required for 4EGI-1-mediated Akt activation. The activated Akt counteracted the anticancer effects of 4EGI-1. In support of this model, inhibition of Akt potentiates the antitumor activity of 4EGI-1 both in vitro and in a xenograft mouse model in vivo. Our results suggest that a combination of 4EGI-1and Akt inhibitor is a rational approach for the treatment of cancer.     

Role of MAP kinase activation in Nramp1 mRNA stability in RAW264.7 macrophages expressing Nramp1(Gly169)

Nramp1 (natural resistance-associated macrophage protein 1) is a phagosomal iron transport molecule. In addition to its anti-microbial activity, Nramp1 exerts a wide range of pleiotropic effects, including increased stability of Nramp1 mRNA and a variety of other mRNA species. Previously, we showed that the increased stability of Nramp1 mRNA is regulated by an oxidant-generated signaling pathway that requires PKC. In the current study, we show that inhibition of ERK1,2 and p38 MAP kinase activities decreases Nramp1 mRNA stability in Mycobacterium avium infected RAW264.7 cells expressing Nramp1(Gly169) but not in RAW264.7-Nramp1(Asp169) cells. Phosphorylation of ERK1,2 and p38 MAP kinases, which could be inhibited by the anti-oxidant BHA and a protein kinase C inhibitor, was higher in M. avium infected RAW264.7-Nramp1(Gly169) cells than in RAW26.47-Nramp1(Asp169) cells. These results suggest that generation of oxidants by Nramp1 iron transport activates MAP kinase signaling cascades that result in stabilization of Nramp1 mRNA.     

Activation of cap-independent translation by variant eukaryotic initiation factor 4G in vivo

Protein synthesis is tightly controlled by assembly of an intricate ribonucleoprotein complex at the m⁷GTP-cap on eukaryotic mRNAs. Ensuing linear scanning of the 5′ untranslated region (UTR) is believed to transfer the preinitiation complex to the initiation codon. Eukaryotic mRNAs are characterized by significant 5′ UTR heterogeneity, raising the possibility of differential control of translation initiation rate at individual mRNAs. Curiously, many mRNAs with unconventional, highly structured 5′ UTRs encode proteins with central biological roles in growth control, metabolism, or stress response. The 5′ UTRs of such mRNAs may influence protein synthesis rate in multiple ways, but most significantly they have been implicated in mediating alternative means of translation initiation. Cap-independent initiation bypasses strict control over the formation of initiation intermediates at the m⁷GTP cap. However, the molecular mechanisms that favor alternative means of ribosome recruitment are not understood. Here we provide evidence that eukaryotic initiation factor (eIF) 4G controls cap-independent translation initiation at the c-myc and vascular endothelial growth factor (VEGF) 5′ UTRs in vivo. Cap-independent translation was investigated in tetracycline-inducible cell lines expressing either full-length eIF4G or a C-terminal fragment (Ct) lacking interaction with eIF4E and poly(A) binding protein. Expression of Ct, but not intact eIF4G, potently stimulated cap-independent initiation at the c-myc/VEGF 5′ UTRs. In vitro RNA-binding assays suggest that stimulation of cap-independent translation initiation by Ct is due to direct association with the c-myc/VEGF 5′ UTR, enabling 43S preinitiation complex recruitment. Our work demonstrates that variant translation initiation factors enable unconventional translation initiation at mRNA subsets with distinct structural features.     

Identification and characterization of a novel splice variant of MuSK

MuSK is a receptor tyrosine kinase that initiates the formation of neuromuscular junctions in response to agrin. Little is known about the ligand-induced activation and kinase-dependent signalling that leads to the clustering of acetylcholine receptors. The ectodomain of these molecule is composed of four Ig-like domains. We describe here the isolation of a novel MuSK splice variant that lacks the third Ig-like domain in its ectodomain. The corresponding RNA is the result of alternative splicing which eliminates two exons. There is 10 times less mRNA for this shorter form than for the long form of MuSK and both forms are regulated coordinately. They decrease strongly after birth and are elevated in denervated muscle. Gene transfer by muscle injection of MuSK DNA into individual muscle fibers demonstrates that kinase-induced acetylcholine receptor clustering caused by overexpression of the two kinases does not depend on the presence of the third Ig-like domain.     

High-dose rapamycin induces apoptosis in human cancer cells by dissociating mTOR complex 1 and suppressing phosphorylation of 4E-BP1

mTOR, the mammalian target of rapamycin, has been widely implicated in signals that promote cell cycle progression and survival in cancer cells. Rapamycin, which inhibits mTOR with high specificity, has consequently attracted much attention as an anti-cancer therapeutic. Rapamycin suppresses phosphorylation of S6 kinase at nano-molar concentrations, however at higher micro-molar doses, rapamycin induces apoptosis in several human cancer cell lines. While much is known about the effect of low dose rapamycin treatment, the mechanistic basis for the apoptotic effects of high-dose rapamycin treatment is not understood. We report here that the apoptotic effects of high-dose rapamycin treatment correlate with suppressing phosphorylation of the mTOR complex 1 substrate, eukaryotic initiation factor 4E (eIF4E) binding protein-1 (4E-BP1). Consistent with this observation, ablation of eIF4E also resulted in apoptorsis in MDA-MB 231 breast cancer cells. We also provide evidence that the differential dose effects of rapamycin are correlated with partial and complete dissociation of Raptor from mTORC1 at low and high doses, respectively. In contrast with MDA-MB-231 cells, MCF-7 breast cancer cells survived rapamycin-induced suppression of 4E-BP1 phosphorylation. We show that survival correlated with a hyper-phosphorylation of Akt at S473 at high rapamycin doses, the suppression of which conferred rapamycin sensitivity. This study reveals that the apoptotic effect of rapamycin requires doses that completely dissociate Raptor from mTORC1 and suppress that phosphorylation of 4E-BP1 and inhibit eIF4E.