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The X-linked CDKL5 gene codes for a kinase whose mutations have been associated with a suite of neurodevelopmental disorders generally characterized by early-onset epileptic encephalopathy and severe intellectual disability. The impact of these mutations on CDKL5 functions and brain development remain mainly unknown, although the importance of maintaining the catalytic activity is generally recognized. Since no cure exists for CDKL5 disorders, the demand for innovative therapies is a real emergency. The recent discovery that CDKL5 is dosage sensitive poses concerns on conventional protein and gene augmentative therapies. Thus, RNA-based therapeutic approaches might be preferred. We studied the efficacy of read-through therapy on CDKL5 premature termination codons (PTCs) that correspond roughly to 15% of all mutations. Our results provide the first demonstration that all tested CDKL5 nonsense mutations are efficiently suppressed by aminoglycoside drugs. The functional characterization of the restored full-length CDKL5 reveals that read-through proteins fully recover their subcellular localization, but only partially rescue their catalytic activity. Since read-through can cause amino acid substitution, CDKL5 patients carrying the PTC outside the catalytic domain might benefit more from a nonsense suppression therapy. Eventually, we demonstrate that non-aminoglycoside drugs, such as Ataluren (PTC124) and GJ072, are unable to induce read-through activity on CDKL5 PTCs. Although these drugs might be more effective in vivo, these results question the validity of the Ataluren phase 2 clinical trial that is currently ongoing on CDKL5 patients.

Accurate chromosome segregation is essential for cell viability. The mitotic spindle is crucial for chromosome segregation, but much remains unknown about factors that regulate spindle assembly. Recent work implicates RNA in promoting proper spindle assembly independently of mRNA translation; however, the mechanism by which RNA performs this function is currently unknown. Here, we show that RNA regulates both the localization and catalytic activity of the mitotic kinase, Aurora-B (AurB), which is present in a ribonucleoprotein (RNP) complex with many mRNAs. Interestingly, AurB kinase activity is reduced in Xenopus egg extracts treated with RNase, and its activity is stimulated in vitro by RNA binding. Spindle assembly defects following RNase-treatment are partially rescued by inhibiting MCAK, a microtubule depolymerase that is inactivated by AurB-dependent phosphorylation. These findings implicate AurB as an important RNA-dependent spindle assembly factor, and demonstrate a translation-independent role for RNA in stimulating AurB.

The p38α, also named Mapk14, is a pro-apoptotic protein, which is reported to be downregulated 2 h after cerebral ischemia. However, little is known what causes the downregulation of p38α protein level. Here, we studied the effect of cerebral ischemia on p38α mRNA expression and p38α protein level in brain of mice. We found that p38α protein level is reduced after middle cerebral artery occlusion. However, at the meantime, p38α mRNA expression has no detectable changes, suggesting that the possible posttranscription is regulated by ischemia. To reveal the mechanism for posttranscription of p38α protein, we tested the effect of miR-128-3p. Using luciferase reporter assay, we found that miR-128-3p could directly target p38α 3'UTR. We further tested the effect of miR-128-3p on the p38α protein level. We found that miR-128-3p strongly decreased the p38α protein level in SH-SY5Y cells after the cells were transfected with miR-128-3p using lentivirus vector containing precursor its RNA sequences. We further found that inhibition of miR-128-3p enhanced the infarct volume of brain in mice. Our study thus confirms that miR-128-3p can downregulate p38α protein level through posttranscription and increase of miR-128-3p level may contribute to neuronal survival in ischemia-induced brain injury.

Scavenger receptor class B, type I (SR-BI), is a high density lipoprotein receptor that mediates the flux of cholesterol between high density lipoprotein and cells. Recent evidence suggests that SR-BI plays a role in atherosclerosis and that inflammatory mediators down-regulate SR-BI in the macrophage. The purpose of this study was to evaluate the ability of lipopolysaccharide (LPS) to down-regulate the activity of the human SR-BI promoter in the macrophage and to delineate the mechanisms involved. Experiments with cultured cells and in vivo derived macrophages showed that LPS has a powerful suppressive effect on SR-BI expression both in vitro and in vivo. Transient transfection studies demonstrated that LPS represses SR-BI promoter activity in the macrophage cell line RAW 264.7. Cotransfection with either a constitutively active p21-activated protein kinase-1 (PAK1) construct (T423E) or a kinase-deficient PAK1 construct (K299R) resulted in repression of the SR-BI promoter, similar to LPS. These results demonstrate that PAK1-mediated down-regulation of the SR-BI promoter is independent of PAK1 kinase activity and suggest that PAK1 mediates the LPS-induced decrease in promoter activity. Cotransfection with constitutively active Cdc42 or Rac expression constructs also resulted in down-regulation of the promoter; whereas the dominant-negative Cdc42 and Rac constructs elevated basal promoter activity and blunted the LPS response. Cotransfection of PAK1 constructs containing mutations in both the kinase domain and the Cdc42/Rac-binding domain attenuated the PAK1-mediated down-regulation of the promoter, suggesting that Rac and Cdc42 are required for PAK1-mediated decreases in SR-BI promoter activity. 5'-Deletion analysis and gel shift data suggest that LPS inhibits binding of a novel transcription factor to a myeloid zing finger protein-1-like element (-476 to -456) in the human SR-BI promoter. These results demonstrate that the PAK1 pathway down-regulates the SR-BI promoter and suggest that activation of this pathway may play an important role in cholesterol trafficking in the vessel wall.

The effects of 5-fluorouracil (FUra) treatment on thymidine kinase (TKase) activity were examined in vivo in CD8F1 mice bearing first generation CD8F1 mouse mammary tumors. TKase activity was not affected by low dose FUra25 (25 mg/kg), a dose which substantially inhibited thymidylate synthase (TSase), but was severely inhibited 24 hr following treatment with FUra100, a weekly maximally tolerated dose, as judged by activity measurements and labeling of DNA with [3H]thymidine. The amount of (FU)RNA was increased markedly with increasing FUra dose from 0.4 nmol/mg DNA at FUra25 to 2.2 nmol/mg DNA at FUra100. At FUra100, TKase activity gradually declined over 24 hr to less than 10% of the control value, remained low for a further 48 hr, and then was gradually restored to control levels by 168 hr. The loss of TKase activity followed the incorporation of FUra into RNA which peaked at 4-5 hr. TKase activity was not restored by removal of endogenous inhibitors but was restored by treatment with uridine. TKase activity was not inhibited by therapeutic levels of methotrexate (300 mg/kg). TKase from murine colon 38 carcinoma was also severely inhibited, but the activity from colon 26 was only partially (50%) inhibited. Ornithine decarboxylase was also inhibited by FUra100 treatment in the CD8F1 tumor. These results demonstrate that certain short-lived, proliferation-related enzymes are affected by FUra doses higher than those required for TSase inhibition, and this effect appears to correlate with incorporation of FUra into RNA. Thus, in some tumors high doses of FUra can inhibit salvage as well as de novo synthesis of thymidylate providing an increased block of DNA synthesis and increased therapeutic advantage.

In the adult I LnJ mouse skeletal muscle, phosphorylase kinase activity is 0.2% of that in normal. This deficiency results from a paucity of mRNA's for the phosphorylase kinase regulatory subunit- α and its isoform α′. However, in the I LnJ neonatal skeletal muscle phosphorylase kinase activity is 20–25% of that in normal. During the first two months of development this activity decreases while in normal tissue it increases. The developmental differences in the magnitude of the I LnJ deficiency indicate the possibility of stage specific mechanisms regulating the accumulation of α α′ mRNAs. To investigate this possibility, the abundance of α α′ mRNAs and of the catalytic subunit, γ, mRNAs were compared by Northern Blot analysis. The results demonstrate that neonatal and adult I LnJ skeletal muscle have a similar paucity of α α′ mRNAs whereas accumulation of γ mRNAs is not significantly different from normal.

Previous studies have shown that the onset of DNA synthesis in Balb/c 3T3 cells appears to be regulated by a labile protein. We have found that induction of thymidine kinase (TK) activity, after quiescent cells are stimulated by the addition of serum, is similarly regulated by a labile protein. Eight hours after serum stimulation, a 6-h pulse of cycloheximide (CHM) caused an excess delay of 2 h in TK induction. A similar delay also was found in the induction of thymidylate synthase (TS). In contrast, the benzo(a)pyrene transformed 3T3 cell line, BP-A31, which had previously been shown to have no excess delay for the onset of DNA synthesis also had no excess delay for the induction of TK activity after a pulse of CHM. The induction of TK was inhibited by actinomycin D and dichlororibofuranosylbenzimidizole (DRB) suggesting a requirement for new RNA synthesis. It did not appear to depend on DNA synthesis as it was not blocked by aphidicolin. In conclusion, the induction of TK activity appears to be regulated by the same labile cellular signal as the onset of DNA synthesis, and to depend on an increase in the level of TK mRNA in late G1 or early S phase.

Isoproterenol stimulates cell proliferation in mouse salivary glands. Prior to the stimulation of DNA synthesis, (3)H-uridine incorporation into RNA is decreased. This decreased incorporation results from a depression of uridylate kinase activity.

Sonic extracts of mouse fibroblast cells actively converted uridine to uridine nucleotides and degraded uridine to uracil. The uridine kinase, but not the uridine phosphorylase activity, varied considerably, depending on the time after subculture of the cells that the enzyme extracts were prepared. The highest levels of uridine kinase activity were observed at about 24 hr after subculture. An increase in enzymatic activity occurred during the lag phase preceding exponential growth. However, between 24 and 72 hr after subculture, the uridine kinase activity declined to about one-third the level at 24 hr. Following vaccinia infection, the uridine kinase activity of mouse fibroblast cells fell sharply. At about 7 hr post inoculation, the enzymatic activity of infected 2-day-old L-M cells decreased to about one third of the activity of uninfected cells. The fall of uridine kinase activity was more pronounced with vaccinia infected 2 and 3-day-old cells than with infected 1-day-old cells. The enzymatic activity of a mixture prepared by adding one volume of a sonic extract from vaccinia infected cells to one volume of a sonic extract from uninfected cells, was approximately equal to the sum of the activities of each of the respective sonicates, thus contraindicating the presence of potent enzyme inhibitors in the extracts from the infected cells. In contrast to the uridine kinase activity, uridine phosphorylase activity of 2-day-old mouse fibroblast cells was not significantly changed following vaccinia infection.

Translation of Abelson murine leukemia (AbLV) 50 S genomic RNA by microinjection into Xenopus laevis oocytes results in synthesis of a protein indistinguishable in tryptic peptide composition, molecular weight, and type C viral structural components from the 120,000 Mr viral-specific polyprotein expressed in AbLV-transformed mink cells. Post-translational cleavage of this polyprotein, designated AbLV P120, can be achieved by the introduction of avian myeloblastosis virus AMV RNA, which encodes an enzyme with specificity for viral polyprotein cleavage sites, to the oocyte translation system. Polyproteins encoded by AbLV, a second transforming isolate of mouse origin designated AK-T8 and feline sarcoma virus (FeSV) are each shown to be highly phosphorylated. Sites of phosphorylation are not restricted to their known phosphorylated structural components, but also include components encoded by their acquired cellular sequences. Immunoprecipitates of AbLV and FeSV polyproteins obtained both from cells and from pseudotype virions, are shown to contain protein kinase activity. Whether this activity is a function of the polyprotein itself or represents a cellular enzyme associated with the viral polyprotein in the form of an enzyme-substrate complex is, as yet, not resolved. By taking advantage of its efficient incorporation into pseudotype virions, AbLV P120 was purified to homogeneity by a combination of agarose gel filtration and ion-exchange chromatography.

Double-stranded RNA dependent kinase R (PKR) is originally identified as an intracellular sensor of viral infection, but its role in bacterial infection remains largely unknown. Here we report that PKR was an important regulator of antibacterial immunity in sepsis. Genetic deletion of PKR or pharmacological inhibition of its kinase activity markedly increased bacterial loads, organ injury, and mortality in polymicrobial infection induced by cecal ligation and puncture (CLP). In contrast, PKR deficiency or inhibition did not affect bacterial loads, organ injury, or mortality when mice were systemically challenged with Escherichia coli, an abundant microbe in the gastrointestinal tract. PKR deficiency or inhibition markedly decreased the release of interleukin (IL)-1β after CLP. Defect in IL-1 signaling phenocopied PKR deficiency or inhibition in CLP-induced bacterial sepsis. Taken together, these findings identified a critical role of the PKR signaling pathway in antibacterial immunity.

The c-fms proto-oncogene is a member of a gene family that has been implicated in tumorigenesis. Glycoproteins encoded by c-fms were identified in cat spleen cells by means of an immune-complex kinase assay performed with monoclonal antibodies to v-fms-coded epitopes. The major form of the normal cellular glycoprotein has an apparent molecular weight of 170,000 and, like the product of the viral oncogene, serves as a substrate for an associated tyrosine-specific protein kinase activity in vitro. The results suggest that the transforming glycoprotein specified by v-fms is a truncated form of a c-fms-coded growth factor receptor.

Background Evidence indicates a cross-regulation between two kinases, leucine-rich repeat kinase 2 (LRRK2) and protein kinase A (PKA). In neurons, LRRK2 negatively regulates PKA activity in spiny projecting neurons during synaptogenesis and in response to dopamine D1 receptor activation acting as an A-anchoring kinase protein (AKAP). In microglia cells, we showed that LRRK2 kinase activity negatively regulates PKA, impacting NF-κB p50 signaling and the inflammatory response. Here, we explore the molecular mechanism underlying the functional interaction between LRRK2 and PKA in microglia. Methods To understand which step of PKA signaling is modulated by LRRK2, we used a combination of in vitro and ex vivo systems with hyperactive or inactive LRRK2 as well as different readouts of PKA signaling. Results We confirmed that LRRK2 kinase activity acts as a negative regulator of PKA activation state in microglia. Specifically, we found that LRRK2 controls PKA by affecting phosphodiesterase 4 (PDE4) activity, modulating cAMP degradation, content, and its dependent signaling. Moreover, we showed that LRRK2 carrying the G2019S pathological mutation downregulates PKA activation causing a reduction of PKA-mediated NF-κB inhibitory signaling, which results, in turn, in increased inflammation in LRRK2 G2019S primary microglia upon α-synuclein pre-formed fibrils priming. Conclusions Overall, our findings indicate that LRRK2 kinase activity is a key regulator of PKA signaling and suggest PDE4 as a putative LRRK2 effector in microglia. In addition, our observations suggest that LRRK2 G2019S may favor the transition of microglia toward an overactive state, which could widely contribute to the progression of the pathology in LRRK2-related PD.

The process of tRNA splicing entails removal of an intron by TSEN (tRNA–splicing endonuclease) and ligation of the resulting exon halves to generate functional tRNAs. In mammalian cells, the RNA kinase CLP1 (cleavage and polyadenylation factor I subunit) associates with TSEN and phosphorylates the 3′ exon at the 5′ end in vitro, suggesting a role for CLP1 in tRNA splicing. Interestingly, recent data suggest that the ATP-binding and/or hydrolysis capacity of CLP1 is required to enhance pre-tRNA cleavage. In vivo, the lack of CLP1 kinase activity leads to progressive motor neuron loss and accumulation of novel 5′ leader–5′ exon tRNA fragments. We have extended the investigation of the biochemical requirements in pre-tRNA splicing and found that β–γ-hydrolysable ATP is crucial for the productive generation of exon halves. In addition, we provide evidence that phosphorylation of the TSEN complex components supports efficient pre-tRNA cleavage. Taken together, our data improve the mechanistic understanding of mammalian pre-tRNA processing and its regulation.

Pharmacological inhibitors of epidermal growth factor receptor (ErbB1) attenuate the ability of CNS myelin to inhibit axonal regeneration. However, it has been claimed that such effects are mediated by off-target interactions. We have tested the role of ErbB1 in axonal regeneration by culturing neurons from ErbB1 knockout mice in the presence of various inhibitors of axonal regeneration: CNS myelin, chondroitin sulfate proteoglycans (CSPG), fibrinogen or polyinosinic:polycytidylic acid (poly I:C). We confirmed that ErbB1 was activated in cultures of cerebellar granule cells exposed to inhibitors of axonal regeneration and that ErbB1 kinase inhibitors promoted neurite outgrowth under these conditions. In the presence of myelin, fibrinogen, CSPG and poly I:C ErbB1 −/− neurons grew longer neurites than neurons expressing ErbB1. Furthermore, inhibitors of ErbB1 kinase did not improve neurite outgrowth from ErbB1 −/− neurons, ruling out an off-target mechanism of action. ErbB1 kinase activity is therefore a valid target for promoting axonal elongation in the presence of many of the molecules believed to contribute to the failure of axonal regeneration in the injured CNS., Highlights ► We test the role of ErbB1 EGFR receptor in controlling neurite outgrowth. ► CNS myelin, CSPGs, fibrinogen and Poly I:C inhibited neurite outgrowth. ► Outgrowth from wild type neurons was disinhibited by ErbB1 kinase inhibitors. ► Disinhibition was shown to be dependent on the presence of ErbB1. ► ErbB1 kinase is a valid target for enhancing axonal regeneration in the CNS.

Activation of p38γ modulates the integrity of the complex formed by the human discs large protein (hDlg) with cytoskeletal proteins, which is important for cell adaptation to changes in environmental osmolarity. Here we report that, in response to hyperosmotic stress, p38γ also regulates formation of complexes between hDlg and the nuclear protein polypyrimidine tract-binding protein-associated-splicing factor (PSF). Following osmotic shock, p38γ in the cell nucleus increases its association with nuclear hDlg, thereby causing dissociation of hDlg-PSF complexes. Moreover, hDlg and PSF bind different RNAs; in response to osmotic shock, p38γ causes hDlg-PSF and hDlg-RNA dissociation independently of its kinase activity. These findings identify a novel nuclear complex and suggest a previously unreported function of p38γ, which is independent of its catalytic activity and could affect mRNA processing and/or gene transcription to aid cell adaptation to osmolarity changes in the environment.

Forkhead L2 (FOXL2) is expressed in the ovary and acts as a transcriptional repressor of the steroidogenic acute regulatory (StAR) gene, a marker of granulosa cell differentiation. Human FOXL2 mutations that produce truncated proteins lacking the COOH terminus result in blepharophimosis/ptosis/epicanthus inversus (BPES) syndrome type I, which is associated with premature ovarian failure (POF). In this study, we investigated whether FOXL2's activity as a transcriptional repressor is regulated by phosphorylation. We found that FOXL2 is phosphorylated at a serine residue and, using yeast two-hybrid screening, identified LATS1 as a potential FOXL2-interacting protein. LATS1 is a serine/threonine kinase whose deletion in mice results in an ovarian phenotype similar to POF. Using coimmunoprecipitation and kinase assays, we confirmed that LATS1 binds to FOXL2 and demonstrated that LATS1 phosphorylates FOXL2 at a serine residue. Moreover, we found that FOXL2 and LATS1 are coexpressed in developing mouse gonads and in granulosa cells of small and medium follicles in the mouse ovary. Last, we demonstrated that coexpression with LATS1 enhances FOXL2's activity as a repressor of the StAR promoter, and this results from the kinase activity of LATS1. These results provide novel evidence that FOXL2 is phosphorylated by LATS1 and that this phosphorylation enhances the transcriptional repression of the StAR gene, a marker of granulosa cell differentiation. These data support our hypothesis that phosphorylation of FOXL2 may be a control mechanism regulating the rate of granulosa cell differentiation and hence, follicle maturation, and its dysregulation may contribute to accelerated follicular development and POF in BPES type I.

The FGFR3 receptor tyrosine kinase represents an attractive target for therapy due to its role in several human disorders including skeletal dysplasias, multiple myeloma, and cervical and bladder carcinomas. By using molecular library screening, we identified a compound named NF449 with inhibitory activity towards FGFR3 signaling. In cultured chondrocytes and murine limb organ culture, NF449 rescued FGFR3-mediated extracellular matrix loss and growth inhibition, which represent two major cellular phenotypes of aberrant FGFR3 signaling in cartilage. Similarly, NF449 antagonized FGFR3 action in the multiple myeloma cell lines OPM2 and KMS11, as evidenced by NF449-mediated reversal of ERK MAP kinase activation and transcript accumulation of CCL3 and CCL4 chemokines, both of which are induced by FGFR3 activation. In cell-free kinase assays, NF449 inhibited the kinase activity of both wild-type and a disease-associated FGFR3 mutant (K650E) in a fashion that appeared noncompetitive with ATP. Our data identify NF449 as a novel antagonist of FGFR3 signaling, useful for FGFR3 inhibition alone or in combination with inhibitors that target the ATP binding site

The RNA-editing enzyme ADAR1 is a double-stranded RNA (dsRNA) binding protein that modifies cellular and viral RNA sequences by adenosine deamination. ADAR1 has been demonstrated to play important roles in embryonic erythropoiesis, viral response, and RNA interference. In human hepatitis virus infection, ADAR1 has been shown to target viral RNA and to suppress viral replication through dsRNA editing. It is not clear whether this antiviral effect of ADAR1 is a common mechanism in response to viral infection. Here, we report a proviral effect of ADAR1 that enhances replication of vesicular stomatitis virus (VSV) through a mechanism independent of dsRNA editing. We demonstrate that ADAR1 interacts with dsRNA-activated protein kinase PKR, inhibits its kinase activity, and suppresses the α subunit of eukaryotic initiation factor 2 (eIF-2α) phosphorylation. Consistent with the inhibitory effect on PKR activation, ADAR1 increases VSV infection in PKR +/+ mouse embryonic fibroblasts; however, no significant effect was found in PKR −/− cells. This proviral effect of ADAR1 requires the N-terminal domains but does not require the deaminase domain. These findings reveal a novel mechanism of ADAR1 that increases host susceptibility to viral infection by inhibiting PKR activation.

The molecules underlying neuron loss in Parkinson's disease (PD) are essentially unknown, and current therapies focus on diminishing symptoms rather than preventing neuron death. We identified RTP801 as a gene whose transcripts were highly induced in a cellular model of PD in which death of neuronal catecholaminergic PC12 cells was triggered by the PD mimetic 6-OHDA. Here, we find that RTP801 protein is also induced in this and additional cellular and animal PD models. To assess the relevance of these observations to PD, we used immunohistochemistry to compare RTP801 expression in postmortem brains from PD and control patients. For all PD brains examined, expression was highly elevated within neuromelanin-containing neurons of the substantia nigra but not in cerebellar neurons. Evaluation of the potential role of RTP801 induction in our cellular model revealed that RTP801 overexpression is sufficient to promote death but does not further elevate death caused by 6-OHDA. Furthermore, RTP801 induction is requisite for death in our cellular PD models and in 6-OHDA-treated cultured sympathetic neurons in that its knockdown by short hairpin RNAs (shRNAs) is protective. The mechanism by which 6-OHDA and RTP801 induce neuron death appears to involve repression of mammalian target of rapamycin (mTOR) kinase activity, and such death is inhibited by shRNAs targeting TSC2 (tuberous sclerosis complex), a protein with which RTP801 interacts to block mTOR activation. Our findings thus suggest that the elevation of RTP801 we detect in PD substantia nigral neurons may mediate their degeneration and death and that RTP801 and its signaling cascade may be novel potential therapeutic targets for the disease.

By sensing viral nucleic acids, host innate receptors elicit signaling pathways converging on TBK1-IFN regulatory factor (IRF)3 axis in mediating IFN-αβ induction and defense mechanisms. In contrast, viruses have evolved with diverse immune evasion/interference mechanisms to undermine innate receptor signaling and IFN response. In this regard, approaches enabling host to overcome such immune evasion/interference mechanisms are urgently needed to combat infections by epidemic/pandemic viruses. In this study, we report that protein kinase CK2 serves as a key component controlling TBK1 and IRF3 activation in IFN-inducing TLR, RIG-I–like receptors, and cGAS/STING signaling pathways. Accordingly, knocking down of CK2 expression or genetic ablation of its kinase activity resulted in elevated IFN-αβ response in response to infection by DNA and RNA viruses. Moreover, PP2A was identified as one of the intermediate phosphatases responsible for CK2-regulated IFN response, suggesting that CK2 may regulate TBK1 and IRF3 activation indirectly. Importantly, blockade of CK2 activity by small molecule inhibitor was able to activate TBK1, whereby eliciting effective host defense mechanisms against hepatitis C virus infection. Taken together, our results identify CK2 as a novel regulator of TBK1 and IRF3 and suggest that targeting CK2 by small molecular inhibitor may be a viable approach to prevent and treat viral infections.

The Eph family of receptor tyrosine kinases regulates numerous biological processes. To examine the biochemical and developmental contributions of specific structural motifs within Eph receptors, wild-type or mutant forms of the EphA4 receptor were ectopically expressed in developing Xenopus embryos. Wild-type EphA4 and a mutant lacking both the SAM domain and PDZ binding motif were constitutively tyrosine phosphorylated in vivo and catalytically active in vitro. EphA4 induced loss of cell adhesion, ventro-lateral protrusions, and severely expanded posterior structures in Xenopus embryos. Moreover, mutation of a conserved SAM domain tyrosine to phenylalanine (Y928F) enhanced the ability of EphA4 to induce these phenotypes, suggesting that the SAM domain may negatively regulate some aspects of EphA4 activity in Xenopus. Analysis of double mutants revealed that the Y928F EphA4 phenotypes were dependent on kinase activity; juxtamembrane sites of tyrosine phosphorylation and SH2 domain-binding were required for cell dissociation, but not for posterior protrusions. The induction of protrusions and expansion of posterior structures is similar to phenotypic effects observed in Xenopus embryos expressing activated FGFR1. Furthermore, the budding ectopic protrusions induced by EphA4 express FGF-8, FGFR1, and FGFR4a. In addition, antisense morpholino oligonucleotide-mediated loss of FGF-8 expression in vivo substantially reduced the phenotypic effects in EphA4Y928F expressing embryos, suggesting a connection between Eph and FGF signaling.

To understand the regulatory activities of kinases in vivo requires their study across a biologically relevant window of activity. To this end, ATP analog-sensitive kinase alleles (ASKAs) specifically sensitive to a competitive inhibitor have been developed. This article tests whether ASKA technology can be applied to complex immunological systems, such as lymphoid development. The results show that when applied to reaggregate thymic organ culture, novel p56Lck ASKAs readily expose a dose-dependent correlation of thymocyte development with a range of p56Lck activity. By regulating kinase activity, rather than amounts of RNA or protein, ASKA technology offers a general means for assessing the quantitative contributions to immunology of numerous kinases emerging from genomics analyses. It can obviate the generation of multiple lines of mice expressing different levels of kinase transgenes and should permit specific biological effects to be associated with defined biochemical activities.

Recent liver regeneration studies indicate that maintaining hepatic Forkhead Box M1B (FoxM1B) expression in 12-month-old (old-aged) Transthyretin-FoxM1B transgenic mice increases hepatocyte proliferation and expression of cell cycle regulatory genes. Because these transgenic CD-1 mice maintain FoxM1B levels during the aging process, we conducted the current study to determine whether adenovirus delivery of the FoxM1B gene (AdFoxM1B) is sufficient to stimulate liver regeneration in old-aged Balb/c mice. Here we show that AdFoxM1B infection of old-aged mice caused a significant increase in FoxM1B expression, hepatocyte DNA replication, and mitosis following partial hepatectomy. This stimulation in hepatocyte S-phase progression was associated with diminished protein expression and perinuclear localization of cyclin-dependent kinase (Cdk) inhibitor p27(Kip1) (p27) protein following partial hepatectomy. In contrast, old-aged mice infected with control virus displayed high hepatocyte levels of p27 protein, which had been localized to the nucleus prior to S-phase. Furthermore, we found that restoring FoxM1B expression did not influence p27 mRNA levels, and this new finding implicates FoxM1B in regulation of p27 protein levels. Likewise, AdFoxM1B-infected regenerating livers displayed elevated S-phase levels of Cdk2 kinase activity compared with old-aged mice infected with control virus. Furthermore, restoring FoxM1B expression in old-aged mice caused elevated levels of Cyclin B1, Cyclin B2, Cdc25B, Cdk1, and p55CDC mRNA as well as stimulating Cdc25B nuclear localization during liver regeneration, all of which are required for mitosis. These studies indicated that an acute delivery of the FoxM1B gene in old-aged mice is sufficient to re-establish proliferation of regenerating hepatocytes, suggesting that FoxM1B can be used for therapeutic intervention to alleviate the reduction in cellular proliferation observed in the elderly.

The Ste20-like kinase, SLK, has diverse cellular functions. SLK mediates organ development, cell cycle progression, cytoskeletal remodeling, cytokinesis, and cell survival. Expression and activity of SLK are enhanced in renal ischemia-reperfusion injury, and overexpression of SLK was shown to induce apoptosis in cultured glomerular epithelial cells (GECs) and renal tubular cells, as well as GEC/podocyte injury in vivo. The SLK protein consists of a N-terminal catalytic domain and an extensive C-terminal domain, which contains coiled-coils. The present study addresses the regulation of SLK activity. Controlled dimerization of the SLK catalytic domain enhanced autophosphorylation of SLK at T183 and S189, which are located in the activation segment. The full-length ectopically- and endogenously-expressed SLK was also autophosphorylated at T183 and S189. Using ezrin as a model SLK substrate (to address exogenous kinase activity), we demonstrate that dimerized SLK 1-373 or full-length SLK can effectively induce activation-specific phosphorylation of ezrin. Mutations in SLK, including T183A, S189A or T193A reduced T183 or S189 autophosphorylation, and showed a greater reduction in ezrin phosphorylation. Mutations in the coiled-coil region of full-length SLK that impair dimerization, in particular I848G, significantly reduced ezrin phosphorylation and tended to reduce autophosphorylation of SLK at T183. In experimental membranous nephropathy in rats, proteinuria and GEC/podocyte injury were associated with increased glomerular SLK activity and ezrin phosphorylation. In conclusion, dimerization via coiled-coils and phosphorylation of T183, S189 and T193 play key roles in the activation and signaling of SLK, and provide targets for novel therapeutic approaches.

Non-coding RNAs are emerging as key players in many fundamental biological processes, including specification of higher-order chromatin structure. We examined the implication of RNA transcribed from mouse centromeric minor satellite repeats in the formation and function of centromere-associated complexes. Here we show that the levels of minor satellite RNA vary during cell-cycle progression, peaking in G2/M phase, concomitant with accumulation of proteins of the chromosomal passenger complex near the centromere. Consistent with this, we describe that murine minor satellite RNA are components of CENP-A-associated centromeric fractions and associate with proteins of the chromosomal passenger complex Aurora B and Survivin at the onset of mitosis. Interactions of endogenous Aurora B with CENP-A and Survivin are sensitive to RNaseA. Likewise, the kinase activity of Aurora B requires an RNA component. More importantly, Aurora B kinase activity can be potentiated by minor satellite RNA. In addition, decreased Aurora B activity after RNA depletion can be specifically rescued by restitution of these transcripts. Together, our data provide new functional evidence for minor satellite transcripts as key partners and regulators of the mitotic kinase Aurora B.

Following exposure to 9-(2-phosphonylmethoxyethyl)adenine (an inhibitor of the cellular DNA polymerases alpha, delta and epsilon), human erythroleukemia K562, human T-lymphoid CEM and murine leukemia L1210 cells markedly accumulated in the S phase of the cell cycle. In contrast to DNA replication, RNA synthesis (transcription) and protein synthesis (mRNA translation) were not affected by 9-(2-phosphonylmethoxyethyl)-adenine. The ribonucleoside triphosphate pools were slightly elevated, while the intracellular levels of all four deoxyribonucleoside triphosphates were 1.5-4-fold increased in 9-(2-phosphonylmethoxyethyl)adenine-treated K562, CEM and L1210 cells. The effect of 9-(2-phosphonylmethoxyethyl)adenine on de novo (thymidylate synthase-mediated) and salvage (thymidine kinase-mediated) dTTP synthesis was investigated using radio-labelled nucleoside precursors. The amount of thymidylate synthase-derived dTTP in the acid soluble pool was 2-4-fold higher in PMEA-treated than in untreated K562 cells, which is in accord with the 3-4-fold expansion of the global dTTP level in the presence of 9-(2-phosphonylmethoxyethyl)adenine. Strikingly, 2-derived dTTP accumulated to a much higher extent (i.e. 16-40-fold) in the soluble dTTP pool upon 9-(2-phosphonylmethoxyethyl)adenine treatment. In keeping with this finding, a markedly increased thymidine kinase activity could be demonstrated in extracts of 9-(2-phosphonylmethoxyethyl)adenine-treated K562 cell cultures. Also, in the presence of 200 microM 9-(2-phosphonylmethoxyethyl)adenine, 14-fold less thymidylate synthase-derived but only 3-fold less thymidine kinase-derived dTTP was incorporated into the DNA of the K562 cells. These data show that thymidine incorporation may be inappropriate as a cell proliferation marker in the presence of DNA synthesis inhibitors such as 9-(2-phosphonylmethoxyethyl)adenine. Our findings indicate that 9-(2-phosphonylmethoxyethyl)adenine causes a peculiar pattern of (deoxy)ribonucleotide metabolism deregulation in drug-treated tumor cells, as a result of the metabolic block imposed by the drug on the S phase of the cell cycle.

Fibulin-5 (FBLN5), an extracellular matrix glycoprotein required for normal elastogenesis, is coordinately expressed with elastin during lung injury and repair. We found that treatment with transforming growth factor-beta (TGF-beta) induced a rapid but transient increase in FBLN5 heterogeneous nuclear RNA (hnRNA) followed by a sustained increased in the steady-state level of FBLN5 mRNA. The transcription start site of the human FBLN5 gene was localized at 221 nucleotides upstream of the translation start site by using primer extension, Northern blots, and functional analysis of transcriptional activity in reporter plasmids containing 5'-flanking regions. TGF-beta markedly increased FBLN5 promoter activity in transient transfection assays. Two putative Smad-binding sites were identified within the proximal promoter and are required for this TGF-beta induction. Electrophoretic gel mobility shift assay revealed that TGF-beta strongly increased binding of Smad2 and Smad3 nuclear complexes to the proximal FBLN5 promoter and induced a Smad2/3-dependent binding of slow migrating nuclear protein complex. FBLN5 mRNA induction by TGF-beta was blocked by pretreatment with TGF-beta receptor inhibitor SB-431542, the phosphatidylinositol 3-kinase (PI3-kinase) inhibitor LY-294002, and actinomycin D. Basal and TGF-beta-induced FBLN5 hnRNA and mRNA were strongly and proportionally decreased by LY-294002, as was TGF-beta-induced phosphorylation of Akt, but not Smad3, as measured by Western blot analysis. In addition, LY-294002 markedly and proportionally decreased FBLN5 promoter activity in transient transfection analyses with TGF-beta-treated or untreated lung fibroblasts. These studies demonstrate that induction of FBLN5 gene expression in lung fibroblasts is mediated via canonical TGF-beta/Smad signaling and requires the PI3-kinase/Akt pathway.

We describe a novel cytoplasmic tyrosine kinase, termed BPK (B cell progenitor kinase), which is expressed in all stages of the B lineage and in myeloid cells. BPK has classic SH1, SH2, and SH3 domains, but lacks myristylation signals and a regulatory phosphorylation site corresponding to tyrosine 527 of c-src. BPK has a long, basic amino-terminal region upstream of the SH3 domain. BPK was evaluated as a candidate for human X-linked agammaglobulinemia (XLA), an inherited immunodeficiency characterized by a severe deficit of B and plasma cells and profound hypogammaglobulinemia. BPK mapped to within 100 kb of a probe defining the polymorphism most closely linked to XLA at DXS178. Reduction in or the absence of BPK mRNA, protein expression, and kinase activity was observed in XLA pre-B and B cell lines. BPK is likely the XLA gene and functions in pathways critical to B cell expansion.

Employing oligonucleotide primers derived from the DNA sequence of rat p70s6k a homologous sequence was shown by polymerase chain reaction (PCR) to be present as a maternal transcript in stage IV-VI Xenopus laevis oocytes. The sequence covered 665 bp of p70s6k and was 97% identical at the amino acid level. When used to probe a Northern blot of the poly(A)+ mRNA from stage VI oocytes, this sequence recognized four transcripts of 1.9, 2.5, 3.2 and 5.2 kb. Specific rat p70s6k antibodies immunoprecipitated active S6 kinase from stage VI oocytes but not unfertilized eggs. The basal level of activity was 3- to 5-fold higher in primed versus non-primed oocytes indicating that p70s6k activation was an early event in maturation. Consistent with this observation, progesterone induced a 10-fold activation of the kinase in non-primed oocytes within 1 h post-induction at a time critical for early activation of protein synthesis. A much smaller, variable peak of activation was observed at 85% germinal vesicle breakdown (GVBD), which was dependent on the rate of maturation. Two members of the pp90rsk family, thought to be the sole S6 kinases present in X.laevis oocytes, exhibited distinct kinetics of activation. Finally, the S6 kinase activity present 1 h post-progesterone stimulation was purified and shown to have a Mr of 70K.

The regulation of mRNA transport is a fundamental process for cytoplasmic sorting of transcripts and spatially controlled translational derepression once properly localized. There is growing evidence that translation is locally modulated as a result of specific synaptic inputs. However, the underlying molecular mechanisms that regulate this translational process are just emerging. We show that KIS, a serine/threonine kinase functionally related to microtubule dynamics and axon development, interacts with three proteins found in RNA granules: KIF3A, NonO, and eEF1A. KIS localizes to RNA granules and colocalizes with the KIF3A kinesin and the β-actin mRNA in cultured cortical neurons. In addition, KIS is found associated with KIF3A and 10 RNP-transported mRNAs in brain extracts. The results of knockdown experiments indicate that KIS is required for normal neurite outgrowth. More important, the kinase activity of KIS stimulates 3' untranslated region-dependent local translation in neuritic projections. We propose that KIS is a component of the molecular device that modulates translation in RNA-transporting granules as a result of local signals.

Innate immunity plays a critical role in the control of viral infections. The induction of innate immune responses requires activation of transcription factors. In particular, NF-κB plays an essential role in activating the expression of cytokines involved in innate immunity such as beta interferon (IFN-β) and interleukin-6 (IL-6). However, the mechanisms by which viruses activate NF-κB are poorly defined. Infection by parainfluenza virus 5 (PIV5), a prototypical member of theParamyxoviridaefamily ofMononegavirales, has been shown to activate the expression of IFN-β and IL-6. To examine how PIV5 induces this expression, we have examined the activation of NF-κB by PIV5 proteins. We have found that expression of PIV5 L protein alone is sufficient to activate NF-κB. The L protein of PIV5, the catalytic component of the viral RNA-dependent RNA polymerase, contains six domains that are conserved among all negative-stranded nonsegmented RNA viruses. We have mapped the region that activates NF-κB to the second domain, which is thought to be involved in RNA synthesis. The activation of NF-κB by L requires AKT1, a serine/threonine kinase, since AKT1 small interfering RNA, an AKT inhibitor as well as a dominant-negative mutant of AKT1, blocks this activation. Furthermore, we have found that L interacts with AKT1 and enhances its phosphorylation. We speculate that L may encode AKT1 kinase activity.

c-Jun N-terminal kinase (JNK) contributes to metalloproteinase (MMP) gene expression and joint destruction in inflammatory arthritis. It is phosphorylated by at least two upstream kinases, the mitogen-activated protein kinase kinases (MEK) MKK4 and MKK7, which are, in turn, phosphorylated by MEK kinases (MEKKs). However, the MEKKs that are most relevant to JNK activation in synoviocytes have not been determined. These studies were designed to assess the hierarchy of upstream MEKKs, MEKK1, MEKK2, MEKK3, and transforming growth factor-beta activated kinase (TAK)1, in rheumatoid arthritis (RA). Using either small interfering RNA (siRNA) knockdown or knockout fibroblast-like synoviocytes (FLSs), MEKK1, MEKK2, or MEKK3 deficiency (either alone or in combination) had no effect on IL-1beta-stimulated phospho-JNK (P-JNK) induction or MMP expression. However, TAK1 deficiency significantly decreased P-JNK, P-MKK4 and P-MKK7 induction compared with scrambled control. TAK1 knockdown did not affect p38 activation. Kinase assays showed that TAK1 siRNA significantly suppressed JNK kinase function. In addition, MKK4 and MKK7 kinase activity were significantly decreased in TAK1 deficient FLSs. Electrophoretic mobility shift assays demonstrated a significant decrease in IL-1beta induced AP-1 activation due to TAK1 knockdown. Quantitative PCR showed that TAK1 deficiency significantly decreased IL-1beta-induced MMP3 gene expression and IL-6 protein expression. These results show that TAK1 is a critical pathway for IL-1beta-induced activation of JNK and JNK-regulated gene expression in FLSs. In contrast to other cell lineages, MEKK1, MEKK2, and MEKK3 did not contribute to JNK phosphorylation in FLSs. The data identify TAK1 as a pivotal upstream kinase and potential therapeutic target to modulate synoviocyte activation in RA.

SummaryThe PML tumor suppressor controls key pathways for growth suppression, induction of apoptosis, and cellular senescence. PML loss occurs frequently in human tumors through unknown posttranslational mechanisms. Casein kinase 2 (CK2) is oncogenic and frequently upregulated in human tumors. Here we show that CK2 regulates PML protein levels by promoting its ubiquitin-mediated degradation dependent on direct phosphorylation at Ser517. Consequently, PML mutants that are resistant to CK2 phosphorylation display increased tumor-suppressive functions. In a faithful mouse model of lung cancer, we demonstrate that Pml inactivation leads to increased tumorigenesis. Furthermore, CK2 pharmacological inhibition enhances the PML tumor-suppressive property in vivo. Importantly, we found an inverse correlation between CK2 kinase activity and PML protein levels in human lung cancer-derived cell lines and primary specimens. These data identify a key posttranslational mechanism that controls PML protein levels and provide therapeutic means toward PML restoration through CK2 inhibition.

Genes crucial for cancer development can be mutated via various mechanisms, which may reflect the nature of the mutagen. In thyroid papillary carcinomas, mutations of genes coding for effectors along the MAPK pathway are central for transformation. BRAF point mutation is most common in sporadic tumors. By contrast, radiation-induced tumors are associated with paracentric inversions activating the receptor tyrosine kinases RET and NTRK1. We report here a rearrangement of BRAF via paracentric inversion of chromosome 7q resulting in an in-frame fusion between exons 1-8 of the AKAP9 gene and exons 9-18 of BRAF. The fusion protein contains the protein kinase domain and lacks the autoinhibitory N-terminal portion of BRAF. It has elevated kinase activity and transforms NIH3T3 cells, which provides evidence, for the first time to our knowledge, of in vivo activation of an intracellular effector along the MAPK pathway by recombination. The AKAP9-BRAF fusion was preferentially found in radiation-induced papillary carcinomas developing after a short latency, whereas BRAF point mutations were absent in this group. These data indicate that in thyroid cancer, radiation activates components of the MAPK pathway primarily through chromosomal paracentric inversions, whereas in sporadic forms of the disease, effectors along the same pathway are activated predominantly by point mutations.

In this study, we report that the function of certain mutant VDRs from patients with hereditary HVDRR can at least be partially restored by phosphorylation and hexafluoro 1,25(OH) 2 D 3 analogs. Our study provides new insights into mechanisms involved in enhancement of mutant VDR function. Introduction: 1,25-Dihydroxyvitamin D-resistant rickets (HVDRR) is a rare genetic disorder caused by inactivating mutations in the vitamin D receptor (VDR). In this study, we examined VDR from patients with HVDRR having mutations in the ligand-binding domain (F251C, I268T, H305Q, E420K). We examined methods of restoring transcriptional activity of these mutants and the mechanisms involved. Materials and Methods: Reporter gene transcriptional assays were used to examine the activation of mutant VDRs. Western-blot analysis, glutathione S-transferase (GST) pull-down assays, and chromatin immunopre- cipitation (ChIP) assays were also used in this study. Results: Using mutant VDRs, H305Q, F251C, I268T, and 10 �8 M 1,25(OH)2D3, only 10-30% of the activity of wildtype (WT) VDR in activating 24(OH)ase transcription was observed. The transcriptional response of mutant VDR mutants was significantly enhanced 2- to 3-fold by co-treatment of VDR mutant transfected COS-7 cells with 1,25(OH)2D3 and okadaic acid (OA; inhibitor of phosphatase; 50 nM). The H305Q mutant was the most responsive (90% of the response exhibited by WT VDR was restored). The E420K mutant was unresponsive to 1,25(OH)2D3 in the presence or absence of OA. The increased transcriptional response correlated with an increase in the interaction between DRIP205 and the mutant VDR. We further provide evidence that OA induces the phosphorylation of CREB-binding protein (CBP), indicating for the first time a correlation between phosphorylation of CBP and enhanced VDR function. Hexafluoro 1,25(OH)2D3 analogs (RO-26-2198 and RO-4383561) also resulted in at least a partial restoration of the transcriptional responsiveness of mutant VDRs I268T, F251C, and H305Q. Our data indicate that the enhanced potency of the hexafluoro analogs may be caused by increased DRIP205 and glucocorticoid receptor interacting protein 1 (GRIP-1) binding to VDRs and enhanced association of VDRs with DNA, as suggested by results of ChIP assays. Conclusion: Our study provides new insights into the mechanisms involved in the enhancement of VDR function by both phosphorylation and hexafluoro analogs and forms a basis for future study of vitamin D analogs or specifically designed kinase activity mediators as potential therapy for the treatment of selected patients with HVDRR. J Bone Miner Res 2005;20:1680-1691. Published online on April 27, 2005; doi: 10.1359/JBMR.050410

Loss of p53 sensitizes to antimicrotubule agents in human tumor cells, but little is known about its role during mitosis. We have identified the Polo-like kinase family member serum inducible kinase (Snk/Plk2) as a novel p53 target gene. Snk/Plk2 mutagenesis demonstrated that its kinase activity is negatively regulated by its C terminus. Small interfering RNA (siRNA)-mediated Snk/Plk2 silencing in the presence of the mitotic poisons paclitaxel (Taxol) or nocodazole significantly increased apoptosis, similar to p53 mutations, which confer paclitaxel sensitivity. Furthermore, we have demonstrated that the apoptosis due to silencing of Snk/Plk2 in the face of spindle damage occurs in mitotic cells and not in cells that have progressed to a G(1)-like state without dividing. Since siRNA directed against Snk/Plk2 promoted death of paclitaxel-treated cells in mitosis, we envision a mitotic checkpoint wherein p53-dependent activation of Snk/Plk2 prevents mitotic catastrophe following spindle damage. Finally, these studies suggest that disruption of Snk/Plk2 may be of therapeutic value in sensitizing paclitaxel-resistant tumors.

The balance of lipid flux in adipocytes is controlled by the opposing actions of lipolysis and lipogenesis, which are controlled primarily by hormone-sensitive lipase and lipoprotein lipase (LPL), respectively. Catecholamines stimulate adipocyte lipolysis through reversible phosphorylation of hormone-sensitive lipase, and simultaneously inhibit LPL activity. However, LPL regulation is complex and previous studies have described translational regulation of LPL in response to catecholamines because of an RNA-binding protein that interacts with the 3'-untranslated region of LPL mRNA. In this study, we identified several protein components of an LPL RNA binding complex. Using an LPL RNA affinity column, we identified two of the RNA-binding proteins as the catalytic (C) subunit of cAMP-dependent protein kinase (PKA), and A kinase anchoring protein (AKAP) 121/149, one of the PKA anchoring proteins, which has known RNA binding activity. To determine whether the C subunit was involved in LPL translation inhibition, the C subunit was depleted from the cytoplasmic extract of epinephrine-stimulated adipocytes by immunoprecipitation. This resulted in the loss of LPL translation inhibition activity of the extract, along with decreased RNA binding activity in a gel shift assay. To demonstrate the importance of the AKAPs, inhibition of PKA-AKAP binding with a peptide competitor (HT31) prevented epinephrine-mediated inhibition of LPL translation. C subunit kinase activity was necessary for LPL RNA binding and translation inhibition, suggesting that the phosphorylation of AKAP121/149 or other proteins was an important part of RNA binding complex formation. The hormonal activation of PKA results in the reversible phosphorylation of hormone-sensitive lipase, which is the primary mediator of adipocyte lipolysis. These studies demonstrate a dual role for PKA to simultaneously inhibit LPL-mediated lipogenesis through inhibition of LPL translation.

nsP3 is one of the four RNA replicase subunits encoded by alphaviruses. The specific essential functions of nsP3 remain unknown, but it is known to be phosphorylated on serine and threonine residues. Here we have completed mapping of the individual phosphorylation sites on Semliki Forest virus nsP3 (482 amino acids) by point mutational analysis of threonine residues. This showed that threonines 344 and 345 represented the major threonine phosphorylation sites in nsP3. Experiments with deletion variants suggested that nsP3 itself had no kinase activity; instead, it was likely to be phosphorylated by multiple cellular kinases. Phosphorylation was not necessary for the peripheral membrane association of nsP3, which was mediated by the N-terminal region preceding the phosphorylation sites. Two deletion variants of nsP3 with either reduced or undetectable phosphorylation were studied in the context of virus infection. Cells infected with mutant viruses produced close to wild type levels of infectious virions; however, the rate of viral RNA synthesis was significantly reduced in the mutants. A virus totally defective in nsP3 phosphorylation and exhibiting a decreased rate of RNA synthesis also exhibited greatly reduced pathogenicity in mice.

Dengue virus (DENV) is the etiologic agent for dengue fever, for which there is no approved vaccine or specific anti-viral drug. As a remedy for this, we explored the use of compounds that interfere with the action of required host factors and describe here the characterization of a kinase inhibitor (SFV785), which has selective effects on NTRK1 and MAPKAPK5 kinase activity, and anti-viral activity on Hepatitis C, DENV and yellow fever viruses. SFV785 inhibited DENV propagation without inhibiting DENV RNA synthesis or translation. The compound did not cause any changes in the cellular distribution of non-structural 3, a protein critical for DENV RNA synthesis, but altered the distribution of the structural envelope protein from a reticulate network to enlarged discrete vesicles, which altered the co-localization with the DENV replication complex. Ultrastructural electron microscopy analyses of DENV-infected SFV785-treated cells showed the presence of viral particles that were distinctly different from viable enveloped virions within enlarged ER cisternae. These viral particles were devoid of the dense nucleocapsid. The secretion of the viral particles was not inhibited by SFV785, however a reduction in the amount of secreted infectious virions, DENV RNA and capsid were observed. Collectively, these observations suggest that SFV785 inhibited the recruitment and assembly of the nucleocapsid in specific ER compartments during the DENV assembly process and hence the production of infectious DENV. SFV785 and derivative compounds could be useful biochemical probes to explore the DENV lifecycle and could also represent a new class of anti-virals.

A molecular cDNA clone (P1 KIN) was isolated that encodes the human RNA-dependent P1/eIF-2 alpha protein kinase. The complete cDNA sequence of the P1 KIN cDNA was determined; the longest open reading frame (ORF) encoded a 551 amino acid protein with a deduced molecular weight of 62055 Da. Transcripts prepared from the P1 KIN cDNA by transcription in vitro with T7 RNA polymerase programmed the cell-free synthesis of a protein indistinguishable by immunoprecipitation and immunoblot gel analyses from the authentic 67-kDa P1 protein synthesized in human U cells treated with interferon (IFN). Furthermore, by use of a sensitive primer extension assay with T7 DNA polymerase, the major site of translation initiation within the deduced ORF of the P1 KIN cDNA was directly identified. Northern RNA gel-blot analysis revealed that the P1 KIN cDNA strongly hybridized to two IFN-induced mRNAs present in both human amnion U cells and HeLa cells; their sizes were 2.5 and 6 kb. Both transcripts were efficiently induced by IFN-alpha, but poorly by IFN-gamma. Polyclonal antibody was prepared against the product of the P1 KIN cDNA expressed in Escherichia coli. In Western blot analysis the antibody recognized a 67-kDa protein induced in human cells by IFN-alpha and, in addition, a 90-kDa protein whose level was not greatly altered by IFN treatment. The IFN-induced 67-kDa protein was found associated with the ribosomal salt-wash fraction of IFN-treated human cells, whereas the 90-kDa protein was predominantly in the S100 soluble fraction. The time course for the induction by IFN-alpha of RNA-dependent protein P1 kinase activity measured by immunoprecipitation was comparable to the time course for protein P1 induction measured by Western immunoblot analysis. The amino acid sequence of P1/eIF-2 alpha protein kinase deduced from the cDNA was 62% identical with the 518-residue murine TIK kinase and contained, within the carboxy-terminal half of the protein, the motifs commonly conserved among protein-serine/threonine kinases. The amino-terminal half of the P1 protein did not possess conserved kinase motifs, but did show extensive homology with vaccinia virus-predicted protein E3L.

The Escherichia coli gene for homoserine kinase (thrB) has been cloned into a simian-virus-40-based eukaryotic expression vector which also includes a neomycin-resistance gene. Mouse 3T3 cells transfected with this plasmid were selected for resistance and screened for homoserine kinase activity. It has thus been possible to isolate clones which are capable of accumulating homoserine O-phosphate when supplied with homoserine. In broken-cell preparations the kinetic constants for the production of homoserine O-phosphate were similar to those of the wild-type E. coli enzyme. These experiments demonstrate that E. coli homoserine kinase can be expressed in an animal cell and that it can successfully phosphorylate L-homoserine in the intact cell utilizing endogenous ATP.

Hepatocyte growth factor (HGF) is a mitogen for hepatocytes, but it is not clear whether HGF stimulates or inhibits hepatocarcinogenesis. We previously reported that HGF transgenic mice under the metallothionein gene promoter developed benign and malignant liver tumors spontaneously after 17 months of age. To elucidate the role of HGF in hepatocarcinogenesis, diethylnitrosamine (DEN) was administered to HGF transgenic mice. HGF overexpression accelerated DEN-induced hepatocarcinogenesis, often accompanied by abnormal blood vessel formation. In this study, 59% of transgenic males (versus 20% of wild-type males) and 39% of transgenic females (versus 2% of wild-type females) developed either benign or malignant liver tumors by 48 weeks (P

A 5'-hydroxyl RNA kinase from mouse L cells has been partially purified and characterized. The enzyme transfers the gamma-phosphorus from ATP to 5'-hydroxyl termini of RNA much more efficiently than DNA substrates, and is virtually inactive on 3'-CMP. The molecular mass of the predominant kinase activity is estimated to be 93-96 kDa from denaturing and non-denaturing polyacrylamide gel analyses. A minor band of lower molecular mass has been also observed. The enzyme activity requires Mg2+ and is inhibited by both Mn2+ and Zn2+. Antibodies to small nuclear ribonucleoproteins have no effect on this activity.

— Harmine, a β-carboline alkaloid, was found to be an efficient photoactive agent against the DNA-containing murine cytomegalovirus (MCMV) and the RNA containing Sindbis virus, a togavirus, both of which contain membranes. In contrast the host cells themselves were relatively resistant. The MCMV was investigated in more detail. Virus which had been inactivated by harmine plus UVA retained its structural integrity and was fully capable of penetrating host cells, following which the viral genome entered the cell nucleus. The viral genes which normally code for the switch-off in thymidine kinase activity arid for the normal cytopathic effects were not expressed; however the viral geneis responsible for the cytotoxic effect due to high multiplicities of infection was still expressed, indicating that there may be a differential block of some viral genes

Plasmids carrying the herpes simplex virus thymidine kinase (tk) gene were used to transfect thymidine kinase-deficient cells of the mouse fibroblast cell line LM(tk-). Individual cell clones were cultivated in selective hypoxanthine-aminopterin-thymidine medium to determine the number of integrated plasmid copies which was almost always in the range of one to three copies per genome. In contrast, cells transfected with plasmids carrying a promoterless "truncated" tk gene typically contained between 10 and 25 copies per genome. Surprisingly, when the truncated tk gene was transfected together with a simian virus 40 DNA segment, including its transcriptional enhancer, the number of integrated tk gene copies was always low, between one and three copies per genome. We have analyzed the genomic organization of integrated truncated tk genes by blot hybridization of restricted cellular DNA and concluded that integrated units of plasmid DNA molecules are arranged in tandem arrays which remain stable in most cases for many cell generations. In only 1 of ca. 20 cell clones did we observe a retraction and expansion of the number of integrated promoterless tk genes as a response to the removal or readdition of selective pressure. Surprisingly, the thymidine kinase activity determined in extracts from cells growing in selective hypoxanthine-aminopterin-thymidine medium (high numbers of integrated tk gene copies) was nearly the same as the enzymatic activity in cells growing in nonselective medium (low copy numbers). Moreover, Northern blots of polyadenylated RNA, extracted from cells growing under selective and nonselective conditions, showed that, in both cases, the major species of tk-specific transcripts was ca. 1.5 kilobases in size, as expected for a tk-specific mRNA containing the entire coding region of the gene. Thus, disproportionate DNA replication appeared not to be essential for an active tk gene expression in these cells. We discuss possible pathways leading to the formation of tandem arrays of integrated truncated tk genes and the conditions required for disproportionate DNA replication in the unique case in which we found a retraction and expansion of tk gene copy numbers as a response to selective growth conditions.