Adrienne S. Gordon

Coordinated Movement of RACK1 with Activated βIIPKC

Protein kinase C (PKC) isozymes move upon activation from one intracellular site to another. PKC-binding proteins, such as receptors for activated C kinase (RACKs), play an important role in regulating the localization and diverse functions of PKC isozymes. RACK1, the receptor for activated βIIPKC, determines the localization and functional activity of βIIPKC. However, the mechanism by which RACK1 localizes activated βIIPKC is not known. Here, we provide evidence that the intracellular localization of RACK1 changes in response to PKC activation. In Chinese hamster ovary cells transfected with the dopamine D2L receptor and in NG108-15 cells, PKC activation by either phorbol ester or a dopamine D2 receptor agonist caused the movement of RACK1. Moreover, PKC activation resulted in thein situ association and movement of RACK1 and βIIPKC to the same intracellular sites. Time course studies indicate that PKC activation induces the association of the two proteins prior to their co-movement. We further show that association of RACK1 and βIIPKC is required for the movement of both proteins. Our results suggest that RACK1 is a PKC shuttling protein that moves βIIPKC from one intracellular site to another.

Ethanol Operant Self‐Administration in Rats Is Regulated by Adenosine A2 Receptors

BACKGROUND Recent findings suggest that adenosine is involved in the neural and behavioral effects of ethanol (EtOH). Studies in neural cell culture show that EtOH, via activation of adenosine A2 receptors, triggers cyclic adenosine monophosphate/protein kinase A (cAMP/PKA) signaling and CRE (cAMP regulatory element)-mediated gene expression and that this effect is blocked by inhibiting G-protein betagamma subunits. Recently, we reported that expression of a betagamma inhibitor in the nucleus accumbens (NAc) reduces EtOH drinking in rats. The NAc expresses high levels of the adenosine A2A receptor in GABAergic medium spiny neurons. If the reinforcing effects of EtOH are mediated through an A2 activation of cAMP/PKA signaling via betagamma, then A2 receptor blockade should attenuate EtOH consumption. Here we tested this hypothesis. Because adenosine A2 and dopamine D2 receptors are coexpressed in neurons of the NAc, we compared the effects of A2 blockade with those of D2 receptor blockade. METHODS Male Long-Evans rats were trained to self-administer 10% EtOH in daily 30-min sessions with an active and an inactive lever. Separate groups of rats were given the D2 antagonist eticlopride (0.005, 0.007, and 0.01 mg/kg), the A2 antagonist 3,7-dimethyl-1-propargylxanthine (DMPX; 1, 3, 5, 7, 10, and 20 mg/kg), and the A1 antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX; 0.125, 0.25, and 0.5 mg/kg) by systemic injection. RESULTS Eticlopride dose-dependently reduced EtOH drinking. DMPX showed a bimodal effect: 10 and 20 mg/kg decreased, but 1 mg/kg increased, EtOH consumption. DPCPX was without effect. CONCLUSIONS In support of our hypothesis, the A2 antagonist DMPX attenuated EtOH self-administration. Low doses of the A2 antagonist enhanced EtOH drinking, consistent with the possibility that rats increase EtOH self-administration to overcome partial A2 blockade. The D2 antagonist eticlopride also decreased EtOH self-administration. These data provide the first evidence that pharmacological modulation of adenosine A2 receptors can regulate EtOH consumption in rats.

Ethanol-induced Translocation of cAMP-dependent Protein Kinase to the Nucleus MECHANISM AND FUNCTIONAL CONSEQUENCES

Ethanol induces translocation of the catalytic subunit (Cα) of cAMP-dependent protein kinase (PKA) from the Golgi area to the nucleus in NG108–15 cells. Ethanol also induces translocation of the RIIβ regulatory subunit of PKA to the nucleus; RI and Cβ are not translocated. Nuclear PKA activity in ethanol-treated cells is no longer regulated by cAMP. Gel filtration and immunoprecipitation analysis confirm that ethanol blocks the reassociation of Cα with RII but does not induce dissociation of these subunits. Ethanol also reduces inhibition of Cα by the PKA inhibitor PKI. Pre-incubation of Cα with ethanol decreases phosphorylation of Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) and casein but has no effect on the phosphorylation of highly charged molecules such as histone H1 or protamine. cAMP-response element-binding protein (CREB) phosphorylation by Cα is also increased in ethanol-treated cells. This increase in CREB phosphorylation is inhibited by the PKA antagonist (R p)-cAMPS and by an adenosine receptor antagonist. These results suggest that ethanol affects a cascade of events allowing for sustained nuclear localization of Cα and prolonged CREB phosphorylation. These events may account for ethanol-induced changes in cAMP-dependent gene expression.

Phosphorylation of membrane proteins at a cholinergic synapse

Endogenous membrane protein kinase activity and protein kinase substrates have been found in membrane fractions enriched in the acetylcholine receptor that were prepared from the electric organ of Torpedo californica. Phosphorylation of four polypeptides is stimulated 9-fold by K+. The specific cholinergic ligand, carbachol, inhibited phosphorylation of these four polypeptides by 72% in the presence of 1mM Na+ and 100 mM K+. The 65,000-dalton component of the acetylcholine receptor in the membrane fraction appears to be phosphorylated by the endogenous protein kinase. These results suggest that protein phosphorylation may play an important role in synaptic events at nicotinic cholinergic synapses.

The role of adenosine and adenosine transport in ethanol-induced cellular tolerance and dependence. Possible biologic and genetic markers of alcoholism

Acute exposure to ethanol in culture inhibits adenosine uptake into cells, thereby increasing the concentration of extracellular adenosine. Extracellular adenosine then reacts with adenosine A2 receptors to stimulate intracellular cAMP production. During prolonged exposure to ethanol, the increase in cAMP is followed by the development of heterologous desensitization of receptors coupled to adenylyl cyclase via Gs, the stimulatory GTP-binding protein. Ethanol-induced heterologous desensitization appears to be due to a reduction in mRNA and protein for G alpha s, a subunit of Gs. This is an example of cellular dependence on ethanol. The important implication of these findings is that a selective inhibitory effect of ethanol on adenosine uptake can lead to desensitization of diverse receptors coupled to cAMP production. Such changes could contribute to the pleiotropic effects of ethanol in the brain and other organs. Prolonged exposure to ethanol also alters the nucleoside transport system. While ethanol inhibits adenosine uptake into naive cells, ethanol no longer inhibits adenosine uptake into cells that have adapted to ethanol. This resistance to ethanol inhibition appears to be a form of cellular tolerance to ethanol. Thus, there appears to be a synergism between ethanol-induced heterologous desensitization of receptor-stimulated cAMP production (cellular dependence) and resistance to ethanol inhibition of adenosine uptake (cellular tolerance), because both lead to reduced intracellular levels of cAMP. Our studies on cAMP signal transduction in cell culture are directly relevant to the pathophysiology of human alcoholism. Heterologous desensitization of cAMP production is demonstrable in lymphocytes taken from actively drinking alcoholics; this measurement appears to be a biologic marker of active alcohol consumption. In addition, regulation of adenosine receptor-dependent cAMP production may be altered in patients at risk to develop alcoholism because of genetic factors. Thus, lymphocytes from alcoholics cultured many generations in the absence of ethanol show increased adenosine receptor-dependent cAMP production and increased sensitivity to ethanol-induced heterologous desensitization. These persistent phenotypic abnormalities in cell culture could be used as genetic markers for alcoholism. Studies are under way to test this possibility.

Cloning of a novel isoform of the mouse NBMPR-sensitive equilibrative nucleoside transporter (ENT1) lacking a putative phosphorylation site.

We have isolated a mouse cDNA clone corresponding to a novel isoform of the NBMPR-sensitive equilibrative nucleoside transporter (ENT1). The cDNA contains a 6 bp deletion in the open reading frame that changes the amino acid composition in a consensus casein kinase II (CKII) phosphorylation site at Ser-254. The clone containing Ser-254 is termed mENT1.1 and the clone lacking the serine termed mENT1.2. The deduced amino acid sequence of mENT1.1 corresponds to the previously cloned human and rat ENT1 proteins at Ser-254. Tissue distribution studies show that mRNA for both ENT1 isoforms are ubiquitously co-expressed in mouse. Analysis of genomic DNA corresponding to mouse ENT1 indicates the isoforms can be produced by alternative splicing at the end of exon 7. CEM/C19 cells stably expressing mENT1.1 and mENT1.2 show similar dose response curves for NBMPR and dipyridamole inhibition of [(3)H]adenosine uptake as well as exhibiting comparable selectivity for both purine and pyrimidine nucleosides but not the corresponding nucleobases.

Identification of a Mitochondrial Phosphoprotein in Brain Synaptic Membrane Preparations

Abstract: A 41,000‐dalton phosphoprotein in crude synaptosomal membrane fractions is characterized by its unique divalent and monovalent cation regulation. It is identified by two‐dimensional gel electrophoresis as the phosphoprotein whose phosphorylation is enhanced by repetitive electrical stimulation of hippocampal brain slices. After sucrose‐gradient ultracentrifugation, this phosphoprotein is found in the mitochondrial subfraction. This suggests that the electrically produced changes in the level of phosphorylation of the 41,000‐dalton polypeptide are probably effects on cellular energetics rather than on specialized neural membrane function.

Protein phosphatase activity in acetylcholine receptor-enriched membranes

Abstract Phosphorylation of the acetylcholine receptor (AChR) ∗ has been demonstrated in AChR-enriched membranes prepared from the electric organ of Torpedo californica . In this report we show that the same AChR-enriched membrane fraction also contains phosphoprotein phosphatase activity which dephosphorylates both the endogenous AChR and exogenous phosphorylated casein. Release of [32P] PO4 from phosphorylated casein was shown to be inhibited by F− as well as GTP. cAMP and cGMP were without effect. Dephosphorylation of the membrane-bound AChR was also inhibited by F−. This phosphorylation-dephosphorylation mechanism may play a role in mediating the function of the AChR at the synapse.

Membrane-bound protein kinase activity in acetylcholine receptor-enriched membranes

Membrane protein phosphorylation may be a general regulatory mechanism mediating the response of cells to exogenous metabolic and physical signals. We have determined that the membrane-bound acetylcholine receptor is the major substrate phosphorylated in situ by a nearby membrane protein kinase. Moreover, these same membranes also contain phosphoprotein phosphatase activity which dephosphorylates the membrane-bound receptor. These findings suggest that reversible phosphorylation of the actylcholine receptor may be critical for receptor function at the synapse. Therefore, it is necessary to define the properties of the enzymes which mediate this phosphorylation-dephosphorylation mechanism. In this report we describe the properties of the first component of this system, the membrane-bound protein kinase in receptor-enriched membranes from the electric organ of Torpedo californica. Only ATP is effective as a phosphate donor for this cyclic AMP-independent membrane kinase; GTP does not support phosphorylation of the receptor. Both casein and histone can also be phosphorylated by the membrane protein kinase, but casein is a better substrate. Although phosphorylation of the receptor appears to be regulated by cholinergic ligands and K+, casein phosphorylation is not specifically affected by these agents. Moreover, while phosphorylation of the acetylcholine receptor is maximal in receptor=enriched membranes, casein phosphorylation is similar in all membrane fractions prepared from the electric organ. Taken together, these findings suggest that the membrane protein kinase activity in receptor-enriched membranes is similar to most other membrane kinases. Therefore, the unique characteristics of membrane-bound acetylcholine receptor phosphorylation appear to be determined by the receptor and its availability as a substrate for the membrane kinase.

Investigation of the Naja naja siamensis toxin binding site of the cholinergic receptor protein from Torpedo electric tissue

The acetylcholine receptor protein has been purified from both Electrophonts and Torpedo in many different laboratories [l-4] . The molecule appears to consist of _t least five subunits [5] . SDS polyacrylamide electrophoresis of the purified protein shows two bands indicating the existence of at least two different types of subunits in the protein molecule [ 1,4,5] . Furthermore the number of substrate molecules bound per oligomer is lower than the number of subunits [6,7] . From these data one could conclude that the receptor molecule is composed not only of chemically but also functionally different types of polypeptide chains. In this paper we present evidence which gives experimental support to this conclusion. By covalently binding radioactively labelled Naja naja siamensis toxin to the receptor molecule we have shown that only one of these subunits binds the toxin.