FRS2α is essential for the fibroblast growth factor to regulate the mTOR pathway and autophagy in mouse embryonic fibroblasts

Although the fibroblast growth factor (FGF) signaling axis plays important roles in cell survival, proliferation, and differentiation, the molecular mechanism underlying how the FGF elicits these diverse regulatory signals is not well understood. By using the Frs2α null mouse embryonic fibroblast (MEF) in conjunction with inhibitors to multiple signaling pathways, here we report that the FGF signaling axis activates mTOR via the FGF receptor substrate 2α (FRS2α)-mediated PI3K/Akt pathway, and suppresses autophagy activity in MEFs. In addition, the PI3K/Akt pathway regulated mTOR is crucial for the FGF signaling axis to suppress autophagy in MEFs. Since autophagy has been proposed to play important roles in cell survival, proliferation, and differentiation, the findings suggest a novel mechanism for the FGF signaling axis to transmit regulatory signals to downstream effectors.     

PPARα is essential for retinal lipid metabolism and neuronal survival

Background

Peroxisome proliferator activated receptor-alpha (PPARα) is a ubiquitously expressed nuclear receptor. The role of endogenous PPARα in retinal neuronal homeostasis is unknown. Retinal photoreceptors are the highest energy-consuming cells in the body, requiring abundant energy substrates. PPARα is a known regulator of lipid metabolism, and we hypothesized that it may regulate lipid use for oxidative phosphorylation in energetically demanding retinal neurons.

Results

We found that endogenous PPARα is essential for the maintenance and survival of retinal neurons, with Pparα ^-/- mice developing retinal degeneration first detected at 8 weeks of age. Using extracellular flux analysis, we identified that PPARα mediates retinal utilization of lipids as an energy substrate, and that ablation of PPARα ultimately results in retinal bioenergetic deficiency and neurodegeneration. This may be due to PPARα regulation of lipid transporters, which facilitate the internalization of fatty acids into cell membranes and mitochondria for oxidation and ATP production.

Conclusion

We identify an endogenous role for PPARα in retinal neuronal survival and lipid metabolism, and furthermore underscore the importance of fatty acid oxidation in photoreceptor survival. We also suggest PPARα as a putative therapeutic target for age-related macular degeneration, which may be due in part to decreased mitochondrial efficiency and subsequent energetic deficits.

     

Hydrophobic cavity in C-terminus is essential for hTNF-α trimer conformation

A variety of tumour necrosis factor α (TNF-α) derivatives have been bioengineered to improve antitumour activity and reduce toxicity. The expression of TNF-α in Escherichia coli usually yields a mixture of homotrimers and monomers; however, only the trimer shows antitumour activity. TNF-αD10, a bioengineered hTNF-α derivative, demonstrated 10-fold higher cytotoxicity against tumour cells compared to hTNF-α, but the trimer to monomer ratio was 58:42. In the present study, we investigated the structural differences between the trimer and the monomer of TNF-αD10. We found that the chemical shifts of the C-terminal Trp(114) in the trimer were significantly different from those in the monomer and that the replacement of Trp(114) with different amino acids remarkably reduced the trimer production. Further analysis of the publicly available X-ray crystallographic data for trimeric and monomeric hTNF-α revealed that the conformation of the U-shaped region formed by the fragment Cys(101)-Trp(114) was different between the two forms: a hydrophilic cavity in the monomer and a hydrophobic cavity in the trimer. These findings suggested the potential approaches of molecular and structural modification for future improvement of hTNF-α trimer production.     

Cell cycle control and plant morphogenesis: is there an essential link?

Plant morphogenesis has some interesting features that may have consequences for the regulation of cell division. In particular, the immobility of plant cells implies the necessity for highly accurate controls, in contrast with the flexibility of many developmental processes in animals. An important question in plant development concerns the status of the relationship between plant morphogenesis and cell division. In this review, we discuss the current knowledge of the molecular mechanisms controlling the plant cell cycle and how this could be differentially regulated during plant morphogenesis. The plant genes involved are homologous to those of other higher eukaryotes, suggesting a similar cell cycle machinery. A variety of mechanisms control these genes, reflecting the complexity of internal and environmental signals to which plants should respond. This intricate network requires an upstream control mechanism to function as a failsafe system and to govern cell division and growth to produce the correct plant shape. BioEssays 21:29–37, 1999. © 1999 John Wiley & Sons, Inc.     

Dephosphorylation of eIF2α is essential for protein synthesis increase and cell cycle progression after sea urchin fertilization

The eukaryotic Initiation Factor 2 (eIF2) is a key regulator of protein synthesis in eukaryotic cells, implicated in the initiation step of translation. Fertilization of the sea urchin eggs triggers a rapid increase in protein synthesis activity, which is necessary for the progress into embryonic cell cycles. Here we demonstrate that fertilization triggers eIF2α dephosphorylation, concomitant with an increase in protein synthesis and that induction of the eIF2α phosphorylation is intimately linked with an inhibition of protein synthesis and cell cycle arrest. Using a phospho-mimetic protein microinjected into sea urchin eggs, we showed that dephosphorylation of eIF2α is necessary for protein synthesis activity and cell division progression following fertilization. Our results demonstrate that regulation of eIF2α plays an important role in the protein synthesis rise that occurs during early development following fertilization.     

Interaction between CK2α and CK2β, the subunits of protein kinase CK2: thermodynamic contributions of key residues on the CK2α surface

The protein Ser/Thr kinase CK2 (former name: casein kinase II) exists predominantly as a heterotetrameric holoenzyme composed of two catalytic subunits (CK2α) bound to a dimer of noncatalytic subunits (CK2β). We undertook a study to further understand how these subunits interact to form the tetramer. To this end, we used recombinant, C-terminal truncated forms of human CK2 subunits that are able to form the holoenzyme. We analyzed the interaction thermodynamics between the binding of CK2α and CK2β as well as the impact of changes in temperature, pH, and the ionization enthalpy of the buffer using isothermal titration calorimetry (ITC). With structure-guided alanine scanning mutagenesis we truncated individual side chains in the hydrophobic amino acid cluster located within the CK2α interface to identify experimentally the amino acids that dominate affinity. The ITC results indicate that Leu41 or Phe54 single mutations were most disruptive to binding of CK2β. Additionally, these CK2α mutants retained their kinase activity. Furthermore, the substitution of Leu41 in combination with Phe54 showed that the individual mutations were not additive, suggesting that the cooperative action of both residues played a role. Interestingly, the replacement of Ile69, which has a central position in the interaction surface of CK2α, only had modest effects. The differences between Leu41, Phe54, and Ile69 in interaction relevance correlate with solvent accessibility changes during the transition from unbound to CK2β-bound CK2α. Identifying residues on CK2α that play a key role in CK2α/CK2β interactions is important for the future generation of small molecule drug design.     

Sonic Hedgehog dependent phosphorylation by CK1α and GRK2 is required for ciliary accumulation and activation of smoothened

Hedgehog (Hh) signaling regulates embryonic development and adult tissue homeostasis through the GPCR-like protein Smoothened (Smo), but how vertebrate Smo is activated remains poorly understood. In Drosophila, Hh dependent phosphorylation activates Smo. Whether this is also the case in vertebrates is unclear, owing to the marked sequence divergence between vertebrate and Drosophila Smo (dSmo) and the involvement of primary cilia in vertebrate Hh signaling. Here we demonstrate that mammalian Smo (mSmo) is activated through multi-site phosphorylation of its carboxyl-terminal tail by CK1α and GRK2. Phosphorylation of mSmo induces its active conformation and simultaneously promotes its ciliary accumulation. We demonstrate that graded Hh signals induce increasing levels of mSmo phosphorylation that fine-tune its ciliary localization, conformation, and activity. We show that mSmo phosphorylation is induced by its agonists and oncogenic mutations but is blocked by its antagonist cyclopamine, and efficient mSmo phosphorylation depends on the kinesin-II ciliary motor. Furthermore, we provide evidence that Hh signaling recruits CK1α to initiate mSmo phosphorylation, and phosphorylation further increases the binding of CK1α and GRK2 to mSmo, forming a positive feedback loop that amplifies and/or sustains mSmo phosphorylation. Hence, despite divergence in their primary sequences and their subcellular trafficking, mSmo and dSmo employ analogous mechanisms for their activation.     

Predominance of CK2α over CK2α′ in the mammalian brain

CK2 is a heterotetrameric ubiquitous kinase consisting of two catalytic subunits and two regulatory subunits. The two catalytic subunits, α and α', are highly homologous but differ in their C-terminal regions. It is not known whether CK2α and α' have distinctive substrate specificity, since no α- or α'-specific substrate has been identified. Thus, it is assumed that the two kinase isoforms overlap in their substrate specificity. CK2 protein levels and activity were found to be elevated in the brain when compared to other organs. Here we have studied the protein levels of CK2α and α' isoforms in nine major brain regions. We found that both, CK2α and α', are expressed in all brain regions tested. Whereas CK2α levels do not vary strongly across the regions, CK2α' levels are slightly higher in the cortex and hippocampus than in other regions. Furthermore, we show that CK2α protein levels in the striatum are relatively high when compared to CK2α'. The approximate stoichiometry ratio of CK2α:CK2α' is 8:1. Therefore, one can consider that CK2α levels are predominant in comparison to CK2α' levels throughout the mammalian brain.     

Activation of protein kinase C-α is essential for stimulation of cell proliferation by ceramide 1-phosphate

We previously demonstrated that ceramide-1-phosphate (C1P) stimulates fibroblast and macrophage proliferation, but the mechanisms involved in this action have only been partially described. Here we demonstrate that C1P induces translocation of protein kinase C-alpha (PKC-α) from the soluble to the membrane fraction of bone marrow-derived macrophages. Translocation of this enzyme was accompanied by its phosphorylation on Ser 657 residue. Activation of PKC-α was independent of prior stimulation of phosphatidylinositol-dependent or phosphatidylcholine-dependent phospholipase C activities, but required activation of sphingomyelin synthesis. Inhibition of PKC-α activation also blocked C1P-stimulated macrophage proliferation indicating that this enzyme is essential for the mitogenic effect of C1P.     

Lipid-mediated unfolding of 3β-hydroxysteroid dehydrogenase 2 is essential for steroidogenic activity

For inner mitochondrial membrane (IMM) proteins that do not undergo N-terminal cleavage, the activity may occur in the absence of a receptor present in the mitochondrial membrane. One such protein is human 3β-hydroxysteroid dehydrogenase 2 (3βHSD2), the IMM resident protein responsible for catalyzing two key steps in steroid metabolism: the conversion of pregnenolone to progesterone and dehydroepiandrosterone to androstenedione. Conversion requires that 3βHSD2 serve as both a dehydrogenase and an isomerase. The dual functionality of 3βHSD2 results from a conformational change, but the trigger for this change remains unknown. Using fluorescence resonance energy transfer, we found that 3βHSD2 interacted strongly with a mixture of dipalmitoylphosphatidylglycerol (DPPG) and dipalmitoylphosphatidylcholine (DPPC). 3βHSD2 became less stable when incubated with the individual lipids, as indicated by the decrease in thermal denaturation (T(m)) from 42 to 37 °C. DPPG, alone or in combination with DPPC, led to a decrease in α-helical content without an effect on the β-sheet conformation. With the exception of the 20 N-terminal amino acids, mixed vesicles protected 3βHSD2 from trypsin digestion. However, protein incubated with DPPC was only partially protected. The lipid-mediated unfolding completely supports the model in which a cavity forms between the α-helix and β-sheet. As 3βHSD2 lacks a receptor, opening the conformation may activate the protein.     

Laminin α1 is essential for mouse cerebellar development

Laminin α1 (Lama1), which is a subunit of laminin-1 (laminin-111), a heterotrimeric ECM protein, is essential for embryonic development and promotes neurite outgrowth in culture. Because the deletion of Lama1 causes lethality at early embryonic stages in mice, the in vivo role of Lama1 in neural development and functions has not yet been possible to determine. In this study, we generated conditional Lama1 knockout (Lama1(CKO)) mice in the epiblast lineage using Sox2-Cre mice. These Lama1(CKO) mice survived, but displayed behavioral disorders and impaired formation of the cerebellum. Deficiency of Lama1 in the pial basement membrane of the meninges resulted in defects in the conformation of the meninges. During cerebellar development, Lama1 deficiency also caused a decrease in the proliferation and migration of granule cell precursors, disorganization of Bergmann glial fibers and endfeet, and a transient reduction in the activity of Akt. A marked reduction in numbers of dendritic processes in Purkinje cells was observed in Lama1(CKO) mice. Together, these results indicate that Lama1 is required for cerebellar development and functions.     

CK2α/CK1α chimeras are sensitive to regulation by the CK2β subunit

The purpose of this study was to analyse the frequency and type of mutations in the coding region of androgen receptor (AR) and to determine the role of polymorphisms in the intron 1 of ERalpha, exon 5 of ERbeta, intron 7 of progesterone, exon 7 of the aromatase (CYP19) and exon 9 of VDR genes in the risk of prostate cancer. PCR-RFLP analysis of all above the genes was on 100 prostate cancer patients and an equal number of matching controls. The study also included PCR-SSCP analyses of exons 2-8 of AR gene. The genotype containing -/- allele of ERalpha gene was statistically significant for the risk of prostate cancer pose (OR, 2.70; 95% CI, 1.08-6.70, P = 0.032) Rr genotype of ERbeta gene also have a higher risk (OR, 1.65; 95% CI, 0.52-5.23) for prostate cancer. The Cys allele of CYP19 gene was also associated with statistically significant increased risk of prostate cancer (OR; 2.28, 95% CI, 1.20-4.35, P = 0.012). tt genotype of codon 352 of VDR gene showed an OR of 0.43 for (95% CI, 0.13-1.39) and an OR for Tt genotype was 0.65 (95% CI, 0.36-1.16). Taken together, the results showed that in North Indian population, ERalpha and CYP19 genes may be playing a role in the risk of prostate cancer.     

Protein kinase Cα is a calpain target in cultured embryonic muscle cells

Previously we isolated a -calpain/PKC complex from skeletal muscle which suggested tight interactions between the Ca²⁺-dependent protease and the kinase in this tissue. Our previous studies also underlined the involvement of ubiquitous calpains in muscular fusion and differentiation. In order to precise the relationships between PKC and ubiquitous calpains in muscle cells, the expression of these two enzymes was first examined during myogenesis of embryonic myoblasts in culture.Our results show that calpains and PKC are both present in myotubes and essentially localized in the cytosolic compartment. Moreover, calpains were mainly present after 40 h of cell differentiation concomitantly with a depletion of PKC content in the particulate fraction and the appearance of PKM fragment. These results suggest a possible calpain dependent down-regulation process of PKCa in our model at the time of intense fusion.In our experimental conditions phorbol myristate acetate (PMA) induced a rapid depletion of pkc in the cytosolic fraction and its translocation toward the particulate fraction. Long term exposure of myotubes in the presence of PMA induced down-regulation of PKC, this process being partially blocked by calpain inhibitors (CS peptide and inhibitor II) and antisense oligonucleotides for the two major ubiquitous calpain isoforms (m- and -calpains).Taken together, our findings argue for an involvement of calpains in the differentiation of embryonic myoblasts by limited proteolytic cleavage of PKC.     

IL-12Rβ2 is essential for the development of experimental cerebral malaria

A Th1 response is required for the development of Plasmodium berghei ANKA (PbA)-induced experimental cerebral malaria (ECM). The role of pro-Th1 IL-12 in malaria is complex and controversial. In this study, we addressed the role of IL-12Rβ2 in ECM development. C57BL/6 mice deficient for IL-12Rβ2, IL-12p40, or IL-12p35 were analyzed for ECM development after blood-stage PbA infection in terms of ischemia and blood flow by noninvasive magnetic resonance imaging and angiography, T cell recruitment, and gene expression. Without IL-12Rβ2, no neurologic sign of ECM developed upon PbA infection. Although wild-type mice developed distinct brain microvascular pathology, ECM-resistant, IL-12Rβ2-deficient mice showed unaltered cerebral microcirculation and the absence of ischemia after PbA infection. In contrast, mice deficient for IL-12p40 or IL-12p35 were sensitive to ECM development. The resistance of IL-12Rβ2-deficient mice to ECM correlated with reduced recruitment of activated T cells and impaired overexpression of lymphotoxin-α, TNF-α, and IFN-γ in the brain after PbA infection. Therefore, IL-12Rβ2 signaling is essential for ECM development but independent from IL-12p40 and IL-12p35. We document a novel link between IL-12Rβ2 and lymphotoxin-α, TNF-α, and IFN-γ expression, key cytokines for ECM pathogenesis.     

RIM2α is a molecular scaffold for Zona pellucidanduced acrosome reaction

Dear Editor,
The acrosome reaction （AR） in mamma-lian spermatozoa is a prerequisite for successful fertilization, because it leads to the release of hydrolytic enzymes from the acrosomal vesicle along with the exposure of the oocyte-recognition protein lzumo on the sperm surface （Bianchi et al., 2014）. AR mainly follows the conserved principles of calcium-regulated exocytosis in neurons/ neurosecretory cells,