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Abstract
Phosphatidylinositol ()P 5-kinase (PtdIns(4)P 5-kinase) catalyzes the last step in the synthesis of phosphatidylinositol 4, 5-bisphosphate (PtdIns(4,5)P2). PtdIns(4,5)P2 is a precursor of diacylglycerol and inositol 1,4,5-trisphosphate and is also involved in regulation of actin cytoskeleton remodeling and membrane traffic. To satisfy such varied demands in several aspects of cell physiology, synthesis of PtdIns(4,5)P2 must be stringently regulated. In this paper we describe extraction, purification, and characterization of PtdIns(4)P 5-kinase from the plasma membranes of Schizosaccharomyces pombe. We also provide evidence that PtdIns(4)P 5-kinase is phosphorylated and inactivated by Cki1, the S. pombe homolog of casein kinase I. Phosphorylation by Cki1 in vitro decreases the activity of PtdIns(4)P 5-kinase. In addition, and most importantly, overexpression of Cki1 in S. pombe results in a reduced synthesis of PtdIns(4,5)P2 and in a lower activity of PtdIns(4)P 5-kinase associated with the plasma membrane. These results suggest that PtdIns(4)P 5-kinase is a target of Cki1 in S. pombe and that Cki1 is involved in regulation of PtdIns(4, 5)P2 synthesis by phosphorylating and inactivating PtdIns(4)P 5-kinase.
Highlights
Inositol-containing phospholipids are found in all eukaryotes and constitute 2– 8% of total cellular phospholipids
The type I and not type II PtdIns(4)P 5-kinase is stimulated by phosphatidic acid (15). This stimulation may be essential for resynthesis of PtdIns(4,5)P2 in response to PtdIns(4,5)P2 hydrolysis by phospholipase C and subsequent conversion of DG to phosphatidic acid
In pilot experiments to determine the subcellular distribution of S. pombe PtdIns(4)P 5-kinase, we found that the PtdIns(4)P 5-kinase activity was exclusively associated with particulate fractions, no activity was detectable in soluble fraction
Summary
The pellet fraction in centrifuge tubes was overlaid with the following volumes and concentrations of sucrose (containing 20 mM Tris-HCl, pH 7.5, 1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, and 20 g/ml each of pepstatin, aprotinin, and leupeptin) at 0 °C: 1 ml ϫ 50%, 1 ml ϫ 47.5%, 1.5 ml ϫ 45%, 1.5 ml ϫ 42%, 1.5 ml ϫ 40%, 1 ml ϫ 37.5%, and 1 ml ϫ 30%. The concentrated sample was loaded onto phenyl-Superose HR 5/5 (Pharmacia) column equilibrated in 10 mM Tris-HCl buffer, pH 7.5, containing 1 M NaCl, 1 mM EGTA, and 0.1% 2-mercaptoethanol. Purified PtdIns(4)P 5-kinase (650 ng) was incubated in SDS sample buffer (1% SDS, 10 mM dithiothreitol, 10% glycerol, 20 mM Tris, pH 6.8) for 15 min at room temperature. Marker enzymes for subcellular fractions were assayed as described previously (28, 29)
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