Utilization ofDrosophila eye to probe the functions of two mammalian serine/threonine kinases, Snk and HsHPK

Here we report a quick functional analysis of two mammalian serine/threonine kinases, a serum inducible kinase (Snk) and Homo sapiens hepatoma protein kinase (HsHPK), using Drosophila eye as a model system. We generated transgenic fly lines carrying constructs of both kinases under control of the GAL upstream activating sequence (UAS). Each UAS line was then crossed to a line in which GAL4 expression was driven by one of the following promoters, eyeless (ey), glass or decapentaplegic. Thus, different kinase mutants can be ectopically expressed in a promoter-dependent manner. We observed that the ectopic expression of either the wild-type or active form of Snk driven by the glass promoter resulted in a rough-eye phenotype. Nevertheless, the ectopic expression of HsHPK under the control of the ey promoter resulted in a small-eye phenotype. The results of this study demonstrated that ectopic expression of these two mammalian genes could be achieved by the regulation of Drosophila promoters. In addition, the effects of these ectopically expressed genes on eye development could be an implication of their functions with respect to cell proliferation and differentiation. Thus, Drosophila eye, with the powerful genetic tools and vast information on eye development available, can be a useful system to probe the functions of mammalian genes in the postgenome era.     

Silencing of the novel p53 target gene Snk/Plk2 leads to mitotic catastrophe in paclitaxel (taxol)-exposed cells

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.     

Induction of a cellular enzyme for energy metabolism by transforming domains of adenovirus E1a.

Brain creatine kinase is a major enzyme of cellular energy metabolism. It is overexpressed in a wide range of tumor cell lines and is used as a tumor marker. We reported recently that the promoter of the human gene has a strong sequence similarity to the adenovirus E2E promoter. This similarity suggested that the brain creatine kinase gene may be regulated by the viral activator E1a. Experiments reported here showed that both enzyme activity and mRNA levels were induced by the oncogenic products of the E1a region of adenovirus type 5, but unlike the viral E2E promoter, which is induced predominantly by E1a domain 3, brain creatine kinase induction required domains 1 and 2. These domains are important for transformation and for the association of E1a with the retinoblastoma gene product and other cellular proteins. The induction by an oncogene of a cellular gene for energy metabolism may be of significance for the metabolic events that take place after oncogenic activation.     

Ceramide kinase, a novel lipid kinase. Molecular cloning and functional characterization

Ceramide-1-phosphate is a sphingolipid metabolite that has been implicated in membrane fusion of brain synaptic vesicles and neutrophil phagolysosome formation. Ceramide-1-phosphate can be produced by ATP-dependent ceramide kinase activity, although little is known of this enzyme because it has not yet been highly purified or cloned. Based on sequence homology to sphingosine kinase type 1, we have now cloned a related lipid kinase, human ceramide kinase (hCERK). hCERK encodes a protein of 537 amino acids that has a catalytic region with a high degree of similarity to the diacylglycerol kinase catalytic domain. hCERK also has a putative N-myristoylation site on its NH(2) terminus followed by a pleckstrin homology domain. Membrane but not cytosolic fractions from HEK293 cells transiently transfected with a hCERK expression vector readily phosphorylated ceramide but not sphingosine or other sphingoid bases, diacylglycerol or phosphatidylinositol. This activity was clearly distinguished from those of bacterial or human diacylglycerol kinases. With natural ceramide as a substrate, the enzyme had a pH optimum of 6.0-7.5 and showed Michaelis-Menten kinetics, with K(m) values of 187 and 32 microm for ceramide and ATP, respectively. Northern blot analysis revealed that hCERK mRNA expression was high in the brain, heart, skeletal muscle, kidney, and liver. A BLAST search analysis using the hCERK sequence revealed that putative ceramide kinases (CERKs) exist widely in diverse multicellular organisms including plants, nematodes, insects, and vertebrates. Phylogenetic analysis revealed that CERKs are a new class of lipid kinases that are clearly distinct from sphingosine and diacylglycerol kinases. Cloning of CERK should provide new molecular tools to investigate the physiological functions of ceramide-1-phosphate.     

Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.

The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gadd153, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gadd153 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays of gadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in taxia telangiectasia.