ABSTRACT
Phosphorylated derivatives of phosphatidylinositol (PtdIns) are key components of many signaling cascades. Many isoforms of PtdIns kinases, PtdIns phosphate kinases and phosphatases use these lipids in amazing networks of signaling cascades that are yet to be understood fully. PtdIns 4-kinase(s) phosphorylates PtdIns at the 4th -OH position of inositol head group and are classified in to type II and III PtdIns 4-kinases. While type III PtdIns 4-kinases are implicated in vesicular trafficking, type II PtdIns 4-kinases are suggested to play a role in cell signaling, cytoskeletal rearrangements, cell motility and in microbial pathogenicity. This paper reviews the role of type II PtdIns 4-kinases in cell signaling cascades in health and disease.
Subject(s)
1-Phosphatidylinositol 4-Kinase/metabolism , Cell Adhesion/physiology , Cytoskeleton/metabolism , Models, Biological , Multienzyme Complexes/physiology , Phosphatidylinositols/metabolism , Signal Transduction/physiologyABSTRACT
<p><b>OBJECTIVE</b>To construct a phosphatidylinositol 4-kinase beta (PI4K-beta) mutant with the 325th to 373rd amino acid codons deleted, and try to develop a simple method for constructing middle fragment deletion mutant.</p><p><b>METHODS</b>In line with the mechanism of gene splicing by overlap extension(SOE), an additional PCR was used to get the PI4K-beta mutant in which the 325th to 373rd amino acid codons were deleted. Then the mutated gene was cloned into pCMV-Tag4A mammalian expression vector.</p><p><b>RESULTS</b>A mutant with the 325th to 373rd amino acid codons deleted was constructed successfully.</p><p><b>CONCLUSION</b>The improved SOE is a very effective and reliable method to construct middle fragment deletion mutant. It is worthy to be popularized.</p>
Subject(s)
1-Phosphatidylinositol 4-Kinase , Genetics , Base Sequence , Genetic Vectors , Genetics , Molecular Sequence Data , Mutagenesis, Site-Directed , Mutation , Genetics , Polymerase Chain Reaction , Methods , Protein Engineering , Methods , Recombinant Proteins , Genetics , Sequence DeletionABSTRACT
<p><b>OBJECTIVE</b>To study the changes in activity of phosphatidylinositol 4 kinase (PI 4 kinase), phosphatidylinositol 4 phosphate 5 kinase (PIP 5 kinase) and protein kinase C (PKC) during myocardial ischemia and elucidate the relationship between phosphatidylinositol signal pathways and prolonged myocardial ischemia.</p><p><b>METHODS</b>In vivo an ischemic rat model was used. Activity of PI 4 kinase, PIP 5 kinase and PKC were measured at different times in postischemic heart cells using isotope analysis.</p><p><b>RESULTS</b>The activity of PI kinase, PIP kinase and PKC in the myocardium increased to peak at 1 hour postischemia, with activities 6.1, 3.0 and 4.0 fold over control levels, respectively. Their activities declined to normal levels with time.</p><p><b>CONCLUSION</b>The phosphatidylinositol signal pathway is involved in prolonged myocardial ischemia, but its mechanism needs further study.</p>
Subject(s)
Animals , Male , Rats , 1-Phosphatidylinositol 4-Kinase , Metabolism , Myocardial Ischemia , Phosphotransferases (Alcohol Group Acceptor) , Metabolism , Protein Kinase C , Metabolism , Random Allocation , Rats, Wistar , Signal TransductionABSTRACT
A PtdIns 4-kinase from rat spleen particulate fraction was purified to homogeneity and its molecular properties were compared with a PtdIns 4-kinase from splenic lymphocytes. The enzyme activity was solubilized from spleen particulate fraction with Triton X-100 and chromatographed sequentially on phosphocellulose, DEAE-sephacel, heparin acrylamide and hydroxyapatite columns. The purified enzyme preparation showed a 55 kDa band on SDS-PAGE with silver staining. Renaturation of the enzyme activity from SDS-PAGE showed that it comigrated with the 55 kDa protein. Characterization of the enzyme showed that it was a type II PtdIns 4-kinase. Polyclonal antibodies raised against PtdIns 4-kinase inhibited the enzyme activity in in vitro assays. Analysis of adult rat tissue particulate fractions on immunoblots showed restricted immunoreactivity among PtdIns 4-kinases. However, the immunoreactivity is conserved in lymphoid tissues from mouse to human, suggesting that lymphoid tissue has a distinct PtdIns 4-kinase. Activation of rat splenocytes with Con A showed two fold increase in PtdIns 4-kinase activity. Comparison of PtdIns 4-kinases from spleen and splenic lymphocytes showed identical chromatographic behaviour, molecular mass, immunoreactivity, K(m) values for PtdIns and inhibition by adenosine.
Subject(s)
1-Phosphatidylinositol 4-Kinase/antagonists & inhibitors , Adenosine/pharmacology , Animals , Enzyme Inhibitors/pharmacology , Humans , Immunochemistry , Kinetics , Lymphocytes/enzymology , Mice , Molecular Weight , Rats , Rats, Wistar , Spleen/enzymologyABSTRACT
Evidence for heightened capacity for signal transduction in rat hepatoma as well as in human breast and ovarian carcinoma cells as reflected by coordinate increases in PI kinase and PIP kinase in the PI phosphorylation sequence leading to the production of second messengers IP3 and DAG is shown. The linkage of signal transduction enzymes with malignant growth is also seen as MDA-MB- 435 human breast carcinoma or ovarian OVCAR-5 cells express their proliferative capacity in tissue culture in the log phase. In both cases, quercetin inhibit cell proliferation with a decline in PI kinase activity and IP3 levels preceding the growth inhibition seen with quercetin. The elevated steady state activities of PI and PIP kinase indicate a metabolic up-regulation in signal transduction capacity of cancer cells which is down-regulated by quercetin. Since the gain in function manifested in the over-expressed capacity for signal transduction confers selective growth advantage to cancer cells, increased activities of PI and PIP kinases may be considered as sensitive targets for cancer chemotherapy. The potential of quercetin as an interceptor of intracellular signal transduction mechanisms needs to be explored.