The protein kinase CK2 is involved in regulation of circadian rhythms in Arabidopsis

A wide range of processes in plants, including expression of certain genes, is regulated by endogenous circadian rhythms. The circadian clock-associated 1 (CCA1) and the late elongated hypocotyl (LHY) proteins have been shown to be closely associated with clock function in Arabidopsis thaliana. The protein kinase CK2 can interact with and phosphorylate CCA1, but its role in the regulation of the circadian clock remains unknown. Here we show that plants overexpressing CKB3, a regulatory subunit of CK2, display increased CK2 activity and shorter periods of rhythmic expression of CCA1 and LHY. CK2 is also able to interact with and phosphorylate LHY in vitro. Additionally, overexpression of CKB3 shortened the periods of four known circadian clock-controlled genes with different phase angles, demonstrating that many clock outputs are affected. This overexpression also reduced phytochrome induction of an Lhcb gene. Finally, we found that the photoperiodic flowering response, which is influenced by circadian rhythms, was diminished in the transgenic lines, and that the plants flowered earlier on both long-day and short-day photoperiods. These data demonstrate that CK2 is involved in regulation of the circadian clock in Arabidopsis.

The STAR protein QKI-6 is a translational repressor

The signal transduction and activation of RNA (STAR) family of RNA-binding proteins, whose members are evolutionarily conserved from yeast to humans, are important for a number of developmental decisions. For example, in the mouse, quaking proteins (QKI-5, QKI-6, and QKI-7) are essential for embryogenesis and myelination , whereas a closely related protein in Caenorhabditis elegans, germline defective-1 (GLD-1), is necessary for germ-line development. Recently, GLD-1 was found to be a translational repressor that acts through regulatory elements, called TGEs (for tra-2 and GLI elements), present in the 3′ untranslated region of the sex-determining gene tra-2. This gene promotes female development, and repression of tra-2 translation by TGEs is necessary for the male cell fates. The finding that GLD-1 inhibits tra-2 translation raises the possibility that other STAR family members act by a similar mechanism to control gene activity. Here we demonstrate, both in vitro and in vivo, that QKI-6 functions in the same manner as GLD-1 and can specifically bind to TGEs to repress translation of reporter constructs containing TGEs. In addition, expression of QKI-6 in C. elegans wild-type hermaphrodites or in hermaphrodites that are partially masculinized by a loss-of-function mutation in the sex-determining gene tra-3 results in masculinization of somatic tissues, consistent with QKI-6 repressing the activity of tra-2. These results strongly suggest that QKI-6 may control gene activity by operating through TGEs to regulate translation. In addition, our data support the hypothesis that other STAR family members may also be TGE-dependent translational regulators.

A peptide export–import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay

The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export–import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase–prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

CCAAT/enhancer binding protein (C/EBP) sites are required for HIV-1 replication in primary macrophages but not CD4+ T cells

The importance of CCAAT/enhancer binding proteins (C/EBPs) and binding sites for HIV-1 replication in primary macrophages, T cell lines and primary CD4+ T cells was examined. When lines overexpressing the C/EBP dominant-negative protein LIP were infected with HIV-1, replication occurred in Jurkat T cells but not in U937 promonocytes, demonstrating a requirement for C/EBP activators by HIV-1 only in promonocytes. Primary macrophages did not support the replication of HIV-1 harboring mutant C/EBP binding sites in the long terminal repeat but Jurkat, H9 and primary CD4+ T cells supported replication of wild-type and mutant HIV-1 equally well. Thus the requirement for C/EBP sites is also confined to monocyte/macrophages. The requirement for C/EBP proteins and sites identifies the first uniquely macrophage-specific regulatory mechanism for HIV-1 replication.

Hepatitis B virus HBx protein sensitizes cells to apoptotic killing by tumor necrosis factor α

Persistent infection with hepatitis B virus (HBV) is a leading cause of human liver disease and is strongly associated with hepatocellular carcinoma, one of the most prevalent forms of human cancer. Apoptosis (programmed cell death) is an important mediator of chronic liver disease caused by HBV infection. It is demonstrated that the HBV HBx protein acutely sensitizes cells to apoptotic killing when expressed during viral replication in cultured cells and in transfected cells independently of other HBV genes. Cells that were resistant to apoptotic killing by high doses of tumor necrosis factor α (TNFα), a cytokine associated with liver damage during HBV infection, were made sensitive to very low doses of TNFα by HBx. HBx induced apoptosis by prolonged stimulation of N-Myc and the stress-mediated mitogen-activated-protein kinase kinase 1 (MEKK1) pathway but not by up-regulating TNF receptors. Cell killing was blocked by inhibiting HBx stimulation of N-Myc or mitogen-activated-protein kinase kinase 1 using dominant-interfering forms or by retargeting HBx from the cytoplasm to the nucleus, which prevents HBx activation of cytoplasmic signal transduction cascades. Treatment of cells with a mitogenic growth factor produced by many virus-induced tumors impaired induction of apoptosis by HBx and TNFα. These results indicate that HBx might be involved in HBV pathogenesis (liver disease) during virus infection and that enhanced apoptotic killing by HBx and TNFα might select for neoplastic hepatocytes that survive by synthesizing mitogenic growth factors.

Resistance to apoptosis caused by PIG-A gene mutations in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is a clonal hematopoietic stem cell disorder resulting from mutations in an X-linked gene, PIG-A, that encodes an enzyme required for the first step in the biosynthesis of glycosylphosphatidylinositol (GPI) anchors. PIG-A mutations result in absent or decreased cell surface expression of all GPI-anchored proteins. Although many of the clinical manifestations (e.g., hemolytic anemia) of the disease can be explained by a deficiency of GPI-anchored complement regulatory proteins such as CD59 and CD55, it is unclear why the PNH clone dominates hematopoiesis and why it is prone to evolve into acute leukemia. We found that PIG-A mutations confer a survival advantage by making cells relatively resistant to apoptotic death. When placed in serum-free medium, granulocytes and affected CD34+ (CD59−) cells from PNH patients survived longer than their normal counterparts. PNH cells were also relatively resistant to apoptosis induced by ionizing irradiation. Replacement of the normal PIG-A gene in PNH cell lines reversed the cellular resistance to apoptosis. Inhibited apoptosis resulting from PIG-A mutations appears to be the principle mechanism by which PNH cells maintain a growth advantage over normal progenitors and could play a role in the propensity of this disease to transform into more aggressive hematologic disorders. These data also suggest that GPI anchors are important in regulating apoptosis.

Heterogeneous nuclear ribonucleoprotein A1 binds to the transcription-regulatory region of mouse hepatitis virus RNA

A cellular protein, previously described as p35/38, binds to the complementary (−)-strand of the leader RNA and intergenic (IG) sequence of mouse hepatitis virus (MHV) RNA. The extent of the binding of this protein to IG sites correlates with the efficiency of the subgenomic mRNA transcription from that IG site, suggesting that it is a requisite transcription factor. We have purified this protein and determined by partial peptide sequencing that it is heterogeneous nuclear ribonucleoprotein (hnRNP) A1, an abundant, primarily nuclear protein. hnRNP A1 shuttles between the nucleus and cytoplasm and plays a role in the regulation of alternative RNA splicing. The MHV(−)-strand leader and IG sequences conform to the consensus binding motifs of hnRNP A1. Recombinant hnRNP A1 bound to these two RNA regions in vitro in a sequence-specific manner. During MHV infection, hnRNP A1 relocalizes from the nucleus to the cytoplasm, where viral replication occurs. These data suggest that hnRNP A1 is a cellular factor that regulates the RNA-dependent RNA transcription of the virus.

Activation of the c-Jun N-terminal kinase pathway by a novel protein kinase related to human germinal center kinase

The c-Jun N-terminal kinase (JNK), or stress-activated protein kinase plays a crucial role in cellular responses stimulated by environmental stress and proinflammatory cytokines. However, the mechanisms that lead to the activation of the JNK pathway have not been elucidated. We have isolated a cDNA encoding a novel protein kinase that has significant sequence similarities to human germinal center kinase (GCK) and human hematopoietic progenitor kinase 1. The novel GCK-like kinase (GLK) has a nucleotide sequence that encodes an ORF of 885 amino acids with 11 kinase subdomains. Endogenous GLK could be activated by UV radiation and proinflammatory cytokine tumor necrosis factor α. When transiently expressed in 293 cells, GLK specifically activated the JNK, but not the p42/44MAPK/extracellular signal-regulated kinase or p38 kinase signaling pathways. Interestingly, deletion of amino acids 353–835 in the putative C-terminal regulatory region, or mutation of Lys-35 in the putative ATP-binding domain, markedly reduced the ability of GLK to activate JNK. This result indicates that both kinase activity and the C-terminal region of GLK are required for maximal activation of JNK. Furthermore, GLK-induced JNK activation could be inhibited by a dominant-negative mutant of mitogen-activated protein kinase kinase kinase 1 (MEKK1) or mitogen-activated protein kinase kinase 4/SAPK/ERK kinase 1 (SEK1), suggesting that GLK may function upstream of MEKK1 in the JNK signaling pathway.

The activation domain of the enhancer binding protein p45NF-E2 interacts with TAFII130 and mediates long-range activation of the α- and β-globin gene loci in an erythroid cell line

We have used the interaction between the erythroid-specific enhancer in hypersensitivity site 2 of the human β-globin locus control region and the globin gene promoters as a paradigm to examine the mechanisms governing promoter/enhancer interactions in this locus. We have demonstrated that enhancer-dependent activation of the globin promoters is dependent on the presence of both a TATA box in the proximal promoter and the binding site for the erythroid-specific heteromeric transcription factor NF-E2 in the enhancer. Mutational analysis of the transcriptionally active component of NF-E2, p45NF-E2, localizes the critical region for this function to a proline-rich transcriptional activation domain in the NH2-terminal 80 amino acids of the protein. In contrast to the wild-type protein, expression of p45 NF-E2 lacking this activation domain in an NF-E2 null cell line fails to support enhancer-dependent transcription in transient assays. More significantly, the mutated protein also fails to reactivate expression of the endogenous β- or α-globin loci in this cell line. Protein-protein interaction studies reveal that this domain of p45 NF-E2 binds specifically to a component of the transcription initiation complex, TATA binding protein associated factor TAFII130. These findings suggest one potential mechanism for direct recruitment of distal regulatory regions of the globin loci to the individual promoters.

Loss of protein kinase C inhibition in the β-T594M variant of the amiloride-sensitive Na+ channel

We previously reported the presence of a novel variant (β-T594M) of the amiloride-sensitive Na+ channel (ASSC) in which the threonine residue at position 594 in the β-subunit has been replaced by a methionine residue. Electrophysiological studies of the ASSC on Epstein–Barr virus (EBV)-transformed lymphocytes carrying this variant showed that the 8-(4-chlorophenylthio) adenosine 3′:5′-cyclic monophosphate (8cpt-cAMP)-induced responses were enhanced when compared to wild-type EBV-transformed lymphocytes. Furthermore, in wild-type EBV-transformed cells, the 8cpt-cAMP-induced response was totally blocked by the phorbol ester, phorbol 12-myristate 13-acetate (PMA). This inhibitory effect of PMA was blocked by a protein kinase C inhibitor, chelerythrine. We now have identified individuals who are homozygous for this variant, and showed that PMA had no effect on the 8cpt-cAMP-induced responses in the EBV-transformed lymphocytes from such individuals. Cells heterozygous for this variant showed mixed responses to PMA, with the majority of cells partially inhibited by PMA. Our results demonstrate that an alteration in a single amino acid residue in the β-subunit of the ASSC can lead to a total loss of inhibition to PMA, and establish the β-subunit as having an important role in conferring a regulatory effect on the ASSC of lymphocytes.

The small GTP-binding protein Rho links G protein-coupled receptors and Gα12 to the serum response element and to cellular transformation

Receptors coupled to heterotrimeric G proteins can effectively stimulate growth promoting pathways in a large variety of cell types, and if persistently activated, these receptors can also behave as dominant-acting oncoproteins. Consistently, activating mutations for G proteins of the Gαs and Gαi2 families were found in human tumors; and members of the Gαq and Gα12 families are fully transforming when expressed in murine fibroblasts. In an effort aimed to elucidate the molecular events involved in proliferative signaling through heterotrimeric G proteins we have focused recently on gene expression regulation. Using NIH 3T3 fibroblasts expressing m1 muscarinic acetylcholine receptors as a model system, we have observed that activation of this transforming G protein-coupled receptors induces the rapid expression of a variety of early responsive genes, including the c-fos protooncogene. One of the c-fos promoter elements, the serum response element (SRE), plays a central regulatory role, and activation of SRE-dependent transcription has been found to be regulated by several proteins, including the serum response factor and the ternary complex factor. With the aid of reporter plasmids for gene expression, we observed here that stimulation of m1 muscarinic acetylcholine receptors potently induced SRE-driven reporter gene activity in NIH 3T3 cells. In these cells, only the Gα12 family of heterotrimeric G protein α subunits strongly induced the SRE, while Gβ1γ2 dimers activated SRE to a more limited extent. Furthermore, our study provides strong evidence that m1, Gα12 and the small GTP-binding protein RhoA are components of a novel signal transduction pathway that leads to the ternary complex factor-independent transcriptional activation of the SRE and to cellular transformation.

Apoptosis and proliferation of dentate gyrus neurons after single and intermittent limbic seizures

Neuronal apoptosis was observed in the rat dentate gyrus in two experimental models of human limbic epilepsy. Five hours after one hippocampal kindling stimulation, a marked increase of in situ terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) of fragmented DNA was observed in nuclei located within and on the hilar border of the granule cell layer and in the polymorphic region. Forty kindling stimulations with 5-min interval produced higher numbers of labeled nuclei compared with one stimulation. The increase of TUNEL-positive nuclei was prevented by the protein synthesis inhibitor cycloheximide but not affected by the N-methyl-d-aspartate receptor antagonist MK-801. Kainic acid-induced seizures lead to a pattern of labeling in the hippocampal formation identical to that evoked by kindling. A large proportion of cells displaying TUNEL-positive nuclei was double-labeled by the neuron-specific antigen NeuN, demonstrating the neuronal identity of apoptotic cells. Either 1 or 40 kindling stimulations also gave rise to a marked increase of the number of cells double-labeled with the mitotic marker bromodeoxyuridine and NeuN in the subgranular zone and on the hilar border of the dentate granule cell layer. The present data show that single and intermittent, brief seizures induce both apoptotic death and proliferation of dentate gyrus neurons. We hypothesize that these processes, occurring early during epileptogenesis, are primary events in the development of hippocampal pathology in animals and possibly also in patients suffering from temporal lobe epilepsy.

Sphingomyelin depletion in cultured cells blocks proteolysis of sterol regulatory element binding proteins at site 1

The current studies explore the mechanism by which the sphingomyelin content of mammalian cells regulates transcription of genes encoding enzymes of cholesterol synthesis. Previous studies by others have shown that depletion of sphingomyelin by treatment with neutral sphingomyelinase causes a fraction of cellular cholesterol to translocate from the plasma membrane to the endoplasmic reticulum where it expands a regulatory pool that leads to down-regulation of cholesterol synthesis and up-regulation of cholesterol esterification. Here we show that sphingomyelinase treatment of cultured Chinese hamster ovary cells prevents the nuclear entry of sterol regulatory element binding protein-2 (SREBP-2), a membrane-bound transcription factor required for transcription of several genes involved in the biosynthesis and uptake of cholesterol. Nuclear entry is blocked because sphingomyelinase treatment inhibits the proteolytic cleavage of SREBP-2 at site 1, thereby preventing release of the active NH2-terminal fragments from cell membranes. Sphingomyelinase treatment thus mimics the inhibitory effect on SREBP processing that occurs when exogenous sterols are added to cells. Sphingomyelinase treatment did not block site 1 proteolysis of SREBP-2 in 25-RA cells, a line of Chinese hamster ovary cells that is resistant to the suppressive effects of sterols, owing to an activating point mutation in the gene encoding SREBP cleavage-activating protein. In 25-RA cells, sphingomyelinase treatment also failed to down-regulate the mRNA for 3-hydroxy-3-methylglutaryl CoA synthase, a cholesterol biosynthetic enzyme whose transcription depends on the cleavage of SREBPs. Considered together with previous data, the current results indicate that cells regulate the balance between cholesterol and sphingomyelin content by regulating the proteolytic cleavage of SREBPs.

D-AKAP2, a novel protein kinase A anchoring protein with a putative RGS domain

Subcellular localization directed by specific A kinase anchoring proteins (AKAPs) is a mechanism for compartmentalization of cAMP-dependent protein kinase (PKA). Using a two-hybrid screen, a novel AKAP was isolated. Because it interacts with both the type I and type II regulatory subunits, it was defined as a dual specific AKAP or D-AKAP1. Here we report the cloning and characterization of another novel cDNA isolated from that screen. This new member of the D-AKAP family, D-AKAP2, also binds both types of regulatory subunits. A message of 5 kb pairs was detected for D-AKAP2 in all embryonic stages and in all adult tissues tested. In brain, skeletal muscle, kidney, and testis, a 10-kb mRNA was identified. In testis, several small mRNAs were observed. Therefore, D-AKAP2 represents a novel family of proteins. cDNA cloning from a mouse testis library identified the full length D-AKAP2. It is composed of 372 amino acids which includes the R binding fragment, residues 333–372, at its C-terminus. Based on coprecipitation assays, the R binding domain interacts with the N-terminal dimerization domain of RIα and RIIα. A putative RGS domain was identified near the N-terminal region of D-AKAP2. The presence of this domain raises the intriguing possibility that D-AKAP2 may interact with a Gα protein thus providing a link between the signaling machinery at the plasma membrane and the downstream kinase.

B cell receptor-associated protein α4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A

Recently, TAP42 was isolated as a high copy suppressor of sit4−, a yeast phosphatase related to protein phosphatase 2A (PP2A). TAP42 is related to the murine α4 protein, which was discovered independently by its association with Ig-α in the B cell receptor complex. Herein we show that a glutathione S-transferase (GST)–α4 fusion protein bound the catalytic subunit (C) of human PP2A from monomeric or multimeric preparations of PP2A in a “pull-down” assay. In an overlay assay, the GST–α4 protein bound to the phosphorylated and unphosphorylated forms of C that were separated in two-dimensional gels and immobilized on filters. The results show direct and exclusive binding of α4 to C. This is unusual because all known regulatory B subunits, or tumor virus antigens, bind stably only to the AC dimer of PP2A. The α4–C form of PP2A had an increased activity ratio compared with the AC form of PP2A when myelin basic protein phosphorylated by mitogen-activated protein kinase and phosphorylase a were used as substrates. Recombinant α4 cleaved from GST was phosphorylated by p56lck tyrosine kinase and protein kinase C. A FLAG-tagged α4 expressed in COS7 cells was recovered as a protein containing phosphoserine and coimmunoprecipitated with the C but not the A subunit of PP2A. Treatment of cells with rapamycin prevented the association of PP2A with FLAG-α4. The results reveal a novel heterodimer α4–C form of PP2A that may be involved in rapamycin-sensitive signaling pathways in mammalian cells.