Ngoc-Diep Vu

Phosphorylation of ATP-Citrate Lyase by Nucleoside Diphosphate Kinase

Rat liver nucleoside diphosphate kinase (NDPK) and PC12 cell cytosol were used to determine whether NDPK could function as a protein kinase. NDPK was phosphorylated on its catalytic histidine using ATP, and the phosphorylated NDPK separated from [γ-32P]ATP. The addition of phosphorylated NDPK to dialyzed PC12 cell cytosol resulted in the phosphorylation of a protein with a subunit molecular mass of about 120 kDa. This phosphorylation appeared to occur by a direct transfer of a phosphoryl group from the catalytic histidine of NDPK to a histidine on the 120-kDa protein. The 120-kDa protein was partially purified and shown by peptide sequencing to be ATP-citrate lyase. ATP-citrate lyase is the primary source of cytosolic acetyl-CoA. NDPK phosphorylated the histidine at the catalytic site of ATP-citrate lyase. This histidine can also be phosphorylated by ATP, and its phosphorylation is the first step in the conversion of citrate and CoA to oxaloacetate and acetyl-CoA by ATP-citrate lyase. The level of phosphorylation of PC12 cell ATP-citrate lyase by phosphorylated NDPK was comparable with that by ATP. Thus, in addition to its nucleoside diphosphate kinase activity, NDPK can function as a protein kinase.

ATP-induced secretion in PC12 cells and photoaffinity labeling of receptors

Abstract— Secretion of catecholamines by rat PC12 cells is strongly stimulated by extracellular ATP via a P2‐type pur‐inergic receptor. ATP‐induced norepinephrine release was inhibited 80% when extracellular Ca2+ was absent. Only four nucleotides, ATP, ATPγS, benzoylbenzoyl ATP (BzATP), and 2‐methylthio‐ATP, gave substantial stimulation of norepinephrine release from PC12 cells. ATP‐induced secretion was inhibited by Mg2+, and this inhibition was overcome by the addition of excess ATP suggesting that ATP4‐was the active ligand. ATP‐induced secretion of catecholamine release was enhanced by treatment of cells with pertussis toxin or 12‐O‐tetradecanoylphorbol 13‐acetate. The stimulatory effects of 12‐O‐tetradecanoyl‐phorbol 13‐acetate and pertussis toxin on norepinephrine release were additive. After brief exposure of intact cells to the photoaffinity analog, [α‐32P]BzATP, two major proteins of 44 and 50 kDa and a minor protein of 97 kDa were labeled. An excess of ATP‐γS and BzATP but not GTP blocked labeling of the proteins by [32P]BzATP. Labeling of the 50‐kDa protein was more sensitive to competition by 2‐methylthio‐ATP than the other labeled proteins, suggesting that the 50‐kDa protein represents the P2 receptor responsible for ATP‐stimulated secretion in these cells.

Phosphorylation of Geranyl and Farnesyl Pyrophosphates by Nm23 Proteins/Nucleoside Diphosphate Kinases

The biochemical mechanism(s) by which Nm23 proteins/nucleoside diphosphate kinases suppress tumor metastasis, inhibit cell motility, and affect cellular differentiation are not known. Here we report that Nm23 proteins can phosphorylate geranyl and farnesyl pyrophosphates to give triphosphates. Wild type Nm23-H1 had higher geranyl and farnesyl pyrophosphate kinase activities than did mutants of Nm23-H1 that do not inhibit cell motility. The phosphorylation of farnesyl pyrophosphate appears to occur in vivo as cells with an elevated level of Nm23-H1 contained more farnesyl triphosphate than did control cells. To our knowledge, this is the first report that farnesyl triphosphate exists in cells. The phosphorylation of farnesyl pyrophosphate by Nm23 proteins could alter isoprenoid metabolism, and cells with an elevated level of Nm23 proteins were found to contain more farnesylated 46- and 24-kDa proteins than did control cells. The phosphorylation of geranyl and farnesyl pyrophosphates by Nm23 proteins provides a novel mechanism by which these proteins might exert their biological effects.

Decreased turnover of phosphatidylinositol accompanies in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers

The in vivo differentiation of embryonic chicken lens epithelial cells into lens fibers is accompanied by a marked decrease in the rate of degradation of phosphatidylinositol. The present experiments were undertaken to determine whether a similar change in phosphatidylinositol metabolism occurs during in vitro lens fiber formation in cultured explants of embryonic chicken lens epithelia. Lens epithelial cells in the explants differentiate into lens fibers following the addition of fetal calf serum, insulin or chicken vitreous humor to the culture medium. The results show that phosphatidylinositol is degraded with a half-life of 3-6 h in cultured lens epithelia that are not stimulated to differentiate. In contrast, no degradation occurs for at least 6 h in lens epithelia stimulated to form lens fibers. The stabilization of phosphatidylinositol is apparent within 4 h after the onset of fiber cell formation, and thus represents an early event in differentiation. The rapid degradation of phosphatidylinositol in lens epithelia is accompanied by comparably rapid synthesis. During this metabolic turnover only the phosphorylinositol portion of the molecule is renewed, as expected if hydrolysis occurs by the action of a phospholipase C, such as phosphatidylinositol phosphodiesterase. Thus, these data suggest that agents which produce in vitro differentiation of embryonic chicken lens epithelial cells into lens fibers lead to a reduction in either the amount or the activity of phospholipase C.

Correlation between phosphatidylinositol degradation and cell division in embryonic chicken lens epithelia

Differentiation of embryonic chicken lens epithelial cells to form lens fibers is associated with a marked decrease in both the rate of phosphatidylinositol degradation and the rate of cell division. In cells of the central region of the lens epithelium, the rate of cell division also declines with developmental age. The present study measures phosphatidylinositol degradation in cultured explants of the central lens epithelium of chicken embryos of different ages to determine the extent of the correlation between phosphatidylinositol degradation and cell division in this tissue. The results show that the rate of phosphatidylinositol degradation also decreases during development and is proportional to the rate of cell division throughout the period from 6 to 19 days of development. Furthermore, stimulating cell division in central explants of lens epithelia of 19-day-old chicken embryos by culturing them in the presence of fetal calf serum produces a proportional increase in the rate of phosphatidylinositol degradation. These findings indicate that cell division and phosphatidylinositol degradation are tightly coupled in this tissue, and raise the possibility that phosphatidylinositol metabolism may regulate some aspect of the cell cycle.

Pertussis toxin modification of PC12 cells inhibits a protein phosphatase 2A-like phosphatase

Abstract: We have found that modification of rat PC12 cells with pertussis toxin resulted in an ∼50% inhibition of a protein phosphatase 2A‐like phosphatase. Protein phosphatase 2A (PP2A) is a major cellular serine/threonine‐specific protein phosphatase. Treatment of extracts from pertussis toxin‐modified PC12 cells with either immobilized alkaline phosphatase or Ca2+ reversed this inhibition. Reactivation of the PP2A‐like phosphatase in Ca2+ appears to result from the dephosphorylation of a protein by the Ca2+/calmodulin‐dependent protein phosphatase calcineurin. The PP2A‐like phosphatase in extracts from pertussis toxin‐modified PC12 cells eluted from a Mono Q column at a higher ionic strength than did the PP2A‐like phosphatase in extracts from control cells. After incubation in Ca2+, the PP2A‐like phosphatase in extracts from pertussis toxin‐modified cells eluted from a Mono Q column at the same ionic strength as did the PP2A‐like phosphatase in extracts from control cells. These results indicate that the effect of pertussis toxin on this PP2A‐like activity results from the phosphorylation of either one of the subunits of the PP2A‐like phosphatase or a protein that when phosphorylated binds to and inhibits this phosphatase. Pertussis toxin modification did not result in the phosphorylation of the catalytic subunit of PP2A. Because phosphorylation regulates the activities of many enzymes and cell surface receptors, a pertussis toxin‐induced decrease in PP2A activity could alter signaling pathways and other cellular processes in which G proteins are not directly involved.

Small membrane-associated gtp-binding proteins of catecholamine-secreting cells

1. Four GTP-binding proteins (23-27 kDa) were identified in membranes from PC12 cells by [alpha 32P]GTP binding to nitrocellulose blots of SDS-polyacrylamide gels. 2. The GTP-binding proteins remained associated with membranes during stimulation of intact cells by K(+)-depolarization or even after addition of Ca2+ to digitonin-permeabilized cells. 3. By two-dimensional gel electrophoresis, six GTP-binding proteins were resolved and based on their mobility, their phosphorylation state appeared independent of Ca2+. 4. Fractionation of PC12 membranes showed that these GTP-binding proteins were broadly distributed in post-nuclear membranes with the plasma membranes containing the highest specific GTP-binding activity. 5. Membrane fractions from bovine adrenal medulla contain similar GTP-binding proteins with GTP-binding intensity also being highest in the plasma membrane. 6. The GTP-binding proteins could be concentrated in the detergent-rich fraction upon Triton X-114 phase separation.

Phosphatidylinositol Degradation is Directly Proportional to the Rate of Cell Division in Embryonic Chicken Lens Epithelia

This study probes the extent of coupling between cell division and phosphatidylinositol (Ptdlns) degradation in embryonic chicken lens epithelial cells. Between 6 and 19 days of development, the rate of cell division in the central region of the lens epithelium declines gradually. By measuring the rate of Ptdlns degradation in cells of this region in chicken embryos aged 6-, 9-, 12- and 19-days, we have found that the rate of Ptdlns degradation is directly proportional to the rate of cell division throughout this developmental period. Furthermore, culturing expiants of the central region of lens epithelia from 19-day-old embryos in the presence of fetal calf serum produces a 3 to 3.5-fold increase in both the rate of cell division and the rate of Ptdlns degradation. These findings demonstrate that Ptdlns degradation and cell division are tightly coupled in this tissue, and raise the possibility that Ptdlns metabolism influences the regulation of the cell cycle.

PC12 Cells as a Model for Neuronal Secretion

PC12 cells are widely used as models for both neuroendocrine secretion and neuronal differentiation. In this article we will briefly review the origin of these cells and the various types of experiments in which they are employed. We will then discuss in some detail their use as a model for neuronal secretion. There are several extensive and authoritative reviews on PC12 cells which provide much more detailed information on the origin, differentiation, and biochemical properties of PC12 cells.1–4