Heterogeneous ribonucleoprotein C displays a repressor activity mediated by T-cell intracellular antigen-1-related/like protein to modulate Fas exon 6 splicing through a mechanism involving Hu antigen R

T-cell intracellular antigen (TIA)-proteins are known regulators of alternative pre-mRNA splicing. In this study, pull-down experiments and mass spectrometry indicate that TIAR/TIAL1 and hnRNP C1/C2 are associated in HeLa nuclear extracts. Co-immunoprecipitation and GST-pull-down assays confirmed this interaction. Interestingly, binding requires the glutamine-rich (Q-rich) C-terminal domain of TIAR and the leucine-rich plus acidic residues-rich C-terminal domains of hnRNP C1/C2. This interaction also occurs in an RNA-dependent manner. Recombinant GFP-TIAR and RFP-hnRNP C1 proteins display partial nuclear co-localization when overexpressed in HeLa cells, and this requires the Q-rich domain of TIAR. hnRNP C1 overexpression in the presence of rate-limiting amounts of TIAR in HeLa and HEK293 cells affects alternative splicing of Fas and FGFR2 minigenes, promoting Fas exon 6 and FGFR2 exon K-SAM skipping, respectively. The repressor activity of hnRNP C1 on Fas exon 6 splicing is mediated by Hu antigen R (HuR). Experiments involving tethering approaches showed that the repressor capacity of hnRNP C1 is associated with an exonic splicing silencer in Fas exon 6. This effect was reversed by splice-site strengthening and is linked to its basic leucine zipper-like motif. These results suggest that hnRNP C1/C2 acts as a bridge between HuR and TIAR to modulate alternative Fas splicing.     

A G protein-coupled receptor with a lipid kinase domain is involved in cell-density sensing

One mechanism multicellular structures use for controlling cell number [1, 2] involves the secretion and sensing of a factor, such as leptin [3] or myostatin [4], in mammals. Dictyostelium cells secrete autocrine factors for sensing cell density prior to aggregation and multicellular development [5, 6] such as CMF (conditioned-medium factor), which enables starving cells to respond to cAMP pulses [7-9]. Its actions are mediated by two receptors. CMFR1 activates a G protein-independent signaling pathway regulating gene expression [10]. An unknown Galpha1-dependent receptor activates phospholipase C (PLC), which regulates the lifetime of Galpha2-GTP [11-13]. Here, we describe RpkA, an unusual seven-transmembrane receptor that is fused to a C-terminal PIP5 kinase domain and that localizes in membranes of a late endosomal compartment. Loss of RpkA resulted in formation of persistent loose aggregates and altered expression of cAMP-regulated genes. The developmental defect can be rescued by full-length RpkA and the transmembrane domain only. The PIP5 kinase domain is dispensable for the developmental role of RpkA. rpkA- cells secrete and bind CMF but are unable to induce downstream responses. Inactivation of Galpha1, a negative regulator of CMF signaling, rescued the developmental defect of the rpkA- cells, suggesting that RpkA actions are mediated by Galpha1.     

A distinct type of alcohol dehydrogenase, adh4+, complements ethanol fermentation in an adh1-deficient strain of Schizosaccharomyces pombe

In the fission yeast Schizosaccharomyces pombe, only one alcohol dehydrogenase gene, adh1(+), has been identified. To elucidate the influence of adh1(+) on ethanol fermentation, we constructed the adh1 null strain (delta adh1). The delta adh1 cells still produced ethanol and grew fermentatively as the wild-type cells. Both DNA microarray and RT-PCR analysis demonstrated that this ethanol production is caused by the enhanced expression of a Saccharomyces cerevisiae ADH4-like gene product (SPAC5H10.06C named adh4(+)). Since the strain lacking both adh1 and adh4 genes (delta adh1 delta adh4) showed non-fermentative retarded growth, only these two ADHs produce ethanol for fermentative growth. This is the first observation that a S. cerevisiae ADH4-like alcohol dehydrogenase functions in yeast ethanol fermentation.     

The novel ankyrin-repeat containing kinase ARCK-1 acts as a suppressor of the Spalten signaling pathway during Dictyostelium development

Spalten (Spn), a member of the PP2C family of Ser/Thr protein phosphatases, is required for Dictyostelium cell-type differentiation and morphogenesis. We have identified a new protein kinase, ARCK-1, through a second site suppressor screen for mutants that allow spn null cells to proceed further through development. ARCK-1 has a C-terminal kinase domain most closely related to Ser/Thr protein kinases and an N-terminal putative regulatory domain with ankyrin repeats, a 14-3-3 binding domain, and a C1 domain, which is required for binding to RasB^GTP in a two-hybrid assay. Disruption of the gene encoding ARCK-1 results in weak, late developmental defects. However, overexpression of ARCK-1 phenocopies the spn null phenotype, consistent with Spn and ARCK-1 being on the same developmental pathway. Our previous analyses of Spn and the present analysis of ARCK-1 suggest a model in which Spn and ARCK-1 differentially control the phosphorylation state of a protein that regulates cell-type differentiation. Dephosphorylation of the substrate by Spn is required for cell-type differentiation. Control of ARCK-1 and Spn activities by upstream signals is proposed to be part of the developmental regulatory program mediating cell-fate decisions in Dictyostelium.     

Direct inhibition of G(1) cdk kinase activity by MyoD promotes myoblast cell cycle withdrawal and terminal differentiation

MyoD has been proposed to facilitate terminal myoblast differentiation by binding to and inhibiting phosphorylation of the retinoblastoma protein (pRb). Here we show that MyoD can interact with cyclin-dependent kinase 4 (cdk4) through a conserved 15 amino acid (aa) domain in the C-terminus of MyoD. MyoD, its C-terminus lacking the basic helix-loop-helix (bHLH) domain, or the 15 aa cdk4-binding domain all inhibit the cdk4-dependent phosphorylation of pRb in vitro. Cellular expression of full-length MyoD or fusion proteins containing either the C-terminus or just the 15 aa cdk4-binding domain of MyoD inhibit cell growth and pRb phosphorylation in vivo. The minimal cdk4-binding domain of MyoD fused to GFP can also induce differentiation of C2C12 muscle cells in growth medium. The defective myogenic phenotype in MyoD-negative BC3H1 cells can be rescued completely only when MyoD contains the cdk4-binding domain. We propose that a regulatory checkpoint in the terminal cell cycle arrest of the myoblast during differentiation involves the modulation of the cyclin D cdk-dependent phosphorylation of pRb through the opposing effects of cyclin D1 and MyoD.     

In vitro binding and expression studies demonstrate a role for the mouse Sry Q-rich domain in sex determination.

The Q-rich domain of the mouse sex determining gene, Sry, is encoded by an in-frame insertion of a repetitive sequence composed of mostly CAG repeats. The exact function of this Q-rich domain is unknown. Studies on the polymorphisms within this Q-rich domain among different domesticus and musculus mouse strains suggest a possible role for this domain in sex determination. Using the farwestern protein-blotting technique and recombinant fusion proteins containing the Sry Q-rich domain as probes, three Sry interactive proteins of 94, 32 and 28 kDa apparent molecular weight (Sip-1, -2 and -3 respectively) were consistently detected in adult testis. Sip expression was detected in somatic cells and was associated with the spermatogenic activity of the testis. During embryogenesis, Sips were readily detected in total tissue extracts of embryos as early as E8.5 day. In fetal gonads of both sexes, their expression peaked around E11.5-13.5 day, at the time of sex determination and differentiation, and decreased drastically towards late stages of gestation. These observations support the hypothesis that the Q-rich domain may contribute to the biological function(s) of mouse Sry through a protein-protein interactive role(s).     

Amyloid of the Candida albicans Ure2p prion domain is infectious and has an in-register parallel β-sheet structure

Ure2p of Candida albicans (Ure2(albicans) or CaUre2p) can be a prion in Saccharomyces cerevisiae, but Ure2p of Candida glabrata (Ure2(glabrata)) cannot, even though the Ure2(glabrata) N-terminal domain is more similar to that of the S. cerevisiae Ure2p (Ure2(cerevisiae)) than Ure2(albicans) is. We show that the N-terminal N/Q-rich prion domain of Ure2(albicans) forms amyloid that is infectious, transmitting [URE3alb] to S. cerevisiae cells expressing only C. albicans Ure2p. Using solid-state nuclear magnetic resonance of selectively labeled C. albicans Ure2p(1-90), we show that this infectious amyloid has an in-register parallel β-sheet structure, like that of the S. cerevisiae Ure2p prion domain and other S. cerevisiae prion amyloids. In contrast, the N/Q-rich N-terminal domain of Ure2(glabrata) does not readily form amyloid, and that formed upon prolonged incubation is not infectious.     

Myogenic differentiation is dependent on both the kinase function and the N-terminal sequence of mammalian target of rapamycin

The mammalian target of rapamycin (mTOR) is a serine/threonine protein kinase known to control initiation of translation through two downstream pathways: eukaryotic initiation factor 4E-binding protein 1 (4E-BP1)/eukaryotic initiation factor 4E and ribosomal p70 S6 kinase (S6K1). We previously showed in C2C12 murine myoblasts that rapamycin arrests cells in G(1) phase and completely inhibits terminal myogenesis. To elucidate the pathways that regulate myogenesis, we established stable C2C12 cell lines that express rapamycin-resistant mTOR mutants (mTORrr; S2035I) that have N-terminal deletions (Delta10 or Delta91) or are full-length kinase-dead mTORrr proteins. Additional clones expressing a constitutively active S6K1 were also studied. Our results show that Delta10mTORrr signals 4E-BP1 and permits rapamycin-treated myoblasts to differentiate, confirming the mTOR dependence of the inhibition of myogenesis by rapamycin. C2C12 cells expressing either Delta91mTORrr or kinase-dead mTORrr(D2338A) could not phosphorylate 4E-BP1 in the presence of rapamycin and could not abrogate the inhibition of myogenesis. Taken together, our results indicate that both the kinase function of mTOR and the N terminus (residues 11-91, containing part of the first HEAT domain) are essential for myogenic differentiation. In contrast, constitutive activation of S6K1 does not abrogate rapamycin inhibition of either proliferation or myogenic differentiation.     

Novel revertants of H-ras oncogene-transformed R6-PKC3 cells.

Rat 6 fibroblasts that overproduce protein kinase C beta 1 (R6-PKC3 cells) are hypersensitive to complete transformation by the T24 H-ras oncogene; yet T24 H-ras-transformed R6-PKC3 cells are killed when exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA) (W.-L. W. Hsiao, G. M. Housey, M. D. Johnson, and I. B. Weinstein, Mol. Cell. Biol. 9:2641-2647, 1989). Treatment of an R6-PKC3 subclone that harbors a T24 H-ras gene under the control of an inducible mouse metallothionein I promoter with ZnSO4 and TPA is extremely cytocidal. This procedure was used to isolate rare revertants that are resistant to this toxicity. Two revertant lines, R-1a and ER-1-2, continue to express very high levels of protein kinase C enzyme activity but, unlike the parental cells, do not grow in soft agar. Furthermore, these revertants are resistant to the induction of anchorage-independent growth by the v-src, v-H-ras, v-raf, and, in the case of the R-1a line, v-fos oncogenes. Both revertant lines, however, retain the ability to undergo morphological alterations when either treated with TPA or infected with a v-H-ras virus, thus dissociating anchorage independence from morphological transformation. The revertant phenotype of both R-1a and ER-1-2 cells is dominant over the transformed phenotype in somatic cell hybridizations. Interestingly, the revertant lines no longer induce the metallothionein I-T24 H-ras construct or the endogenous metallothionein I and II genes in response to three distinct agents: ZnSO4, TPA, and dexamethasone. The reduction in activity of metallothionein promoters seen in these revertants may reflect defects in signal transduction pathways that control the expression of genes mediating specific effects of protein kinase C and certain oncogenes in cell transformation.     

Cloning and characterization of two human VIP1-like inositol hexakisphosphate and diphosphoinositol pentakisphosphate kinases

Eukaryotes possess numerous inositol phosphate (IP) and diphosphoinositol phosphate (PP-IPs or inositol pyrophosphates) species that act as chemical codes important for intracellular signaling pathways. Production of IP and PP-IP molecules occurs through several classes of evolutionarily conserved inositol phosphate kinases. Here we report the characterization of a human inositol hexakisphosphate (IP6) and diphosphoinositol pentakisphosphate (PP-IP5 or IP7) kinase with similarity to the yeast enzyme Vip1, a recently identified IP6/IP7 kinase (Mulugu, S., Bai, W., Fridy, P. C., Bastidas, R. J., Otto, J. C., Dollins, D. E., Haystead, T. A., Ribeiro, A. A., and York, J. D. (2007) Science 316, 106-109). Recombinant human VIP1 exhibits in vitro IP6 and IP7 kinase activities and restores IP7 synthesis when expressed in mutant yeast. Expression of human VIP1 in HEK293T cells engineered to produce high levels of IP7 results in dramatic increases in bisdiphosphoinositol tetrakisphosphate (PP2-IP4 or IP8). Northern blot analysis indicates that human VIP1 is expressed in a variety of tissues and is enriched in skeletal muscle, heart, and brain. The subcellular distribution of tagged human VIP1 is indicative of a cytoplasmic non-membrane localization pattern. We also characterized human and mouse VIP2, an additional gene product with nearly 90% similarity to VIP1 in the kinase domain, and observed both IP6 and IP7 kinase activities. Our data demonstrate that human VIP1 and VIP2 function as IP6 and IP7 kinases that act along with the IP6K/Kcs1-class of kinases to convert IP6 to IP8 in mammalian cells, a process that has been found to occur in response to various stimuli and signaling events.