Repeated amphetamine couples norepinephrine transporter and calcium channel activities in PC12 cells.

Repeated intermittent amphetamine enhances efflux of dopamine through the dopamine transporter in rat basal ganglia and through the norepinephrine transporter in rat pheochromocytoma PC12 cells. Extracellular Ca2+ is required for the detection of this enhancement in the rat. In this study, we examined the role of Ca2+ and Ca2+ channels in the enhanced amphetamine-induced dopamine efflux that develops in PC12 cells following repeated intermittent amphetamine. Repeated pretreatment of PC12 cells with 1 microM amphetamine followed by a drug-free period increased amphetamine-induced efflux of dopamine compared with controls. The enhancement in amphetamine-induced dopamine efflux depended upon the presence of extracellular Ca2+ and was inhibited by the blockade of N-type and L-type Ca2+ channels. The enhanced dopamine efflux was not altered by tetanus toxin or reserpine, treatments that abrogate synaptic vesicle-mediated, exocytotic dopamine efflux. Measurement of intracellular Ca2+ concentrations using fura-2/acetoxymethyl ester revealed that amphetamine increased intracellular Ca2+ by a transporter-dependent mechanism. In amphetamine-pretreated cells, amphetamine elicited a greater increase in intracellular Ca2+; this increase depended upon the presence of extracellular Ca2+ and N- and L-type Ca2+ channel activity. The enhanced amphetamine-induced dopamine efflux requires Ca2+/calmodulin kinase activity. In vehicle-treated cells, 1 microM amphetamine inhibited the calmodulin kinase activity although it did not in amphetamine-pretreated cells. This study suggests that repeated intermittent amphetamine couples norepinephrine transporter activity and Ca2+ signaling.

Alternative splicing of Drosophila calcium/calmodulin-dependent protein kinase II regulates substrate specificity and activation.

Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate multiple isoforms that vary only in a region between the calmodulin-binding domain and the association domain. This variation has been shown to modulate activation of the enzyme by calmodulin. In this study we examine the ability of seven of the Drosophila isoforms to phosphorylate purified protein substrates and to be inhibited by a substrate analog, and the response of six of the isoforms to a mutant form of calmodulin (V91G) that was isolated in a genetic screen. Significant variation in Kms for Eag, a potassium channel, and Adf-1, a transcription factor, were found. In the case of the a peptide inhibitor, AC3I, there were significant variations in Ki between isoforms. Kact for V91G calmodulin was increased for all of the isoforms. In addition, one isoform, RI, exhibited a lower Vmax when assayed with this mutant CaM. These results indicate that the variable domain of calcium/calmodulin-dependent protein kinase II is capable of altering the substrate specificity of the catalytic domain and the activation response to calmodulin.

Functional diversity of alternatively spliced isoforms of Drosophila Ca2+/calmodulin-dependent protein kinase II. A role for the variable domain in activation.

Isoforms of calcium/calmodulin-dependent protein kinase II from Drosophila (R1-R6 and R3A) showed differential activation by two series of mutant calmodulins, B1K-B4K and B1Q-B4Q. These mutant calmodulins were generated by changing a glutamic acid in each of the four calcium binding sites to either glutamine or lysine, altering their calcium binding properties. All mutations produced activation defects, with the binding site 4 and B1Q mutants the most severe. Activation differed substantially between isoforms. R4, R5, and R6 were the least sensitive to mutations in calmodulin, while R1, R3, and R3A were the most sensitive. Activation of R1 and R2 by B4K and activation of R3 and R3A by B2K and B2Q produced significant (6-fold and almost 3-fold, respectively) differences in Kact between isoforms that differ structurally by a single amino acid. These differences could not be accounted for by differential binding, as all isoforms showed almost identical binding patterns with the mutants. High binding affinity did not always correlate with ability to increase enzyme activity, implying that activation occurs in at least two steps. The isoform-specific differences seen in this study reflect a role for the COOH-terminal variable region in activation of CaM kinase II.

Functional heterogeneity of alternatively spliced isoforms of Drosophila Ca2+/calmodulin-dependent protein kinase II.

The gene for Drosophila calcium/calmodulin-dependent protein kinase II is alternatively spliced to generate up to 18 different proteins that vary only in a region analogous to the point where mammalian alpha, beta, gamma, and delta isozymes show the greatest divergence from each other. To investigate the function of this variable region, we have characterized the catalytic and structural properties of six of the Drosophila isoforms. By several criteria (domain organization, low affinity for calmodulin, holoenzyme structure, and ability to autophosphorylate and become independent of calcium), these proteins are functional homologues of the mammalian calcium/calmodulin-dependent protein kinase II. Two major isoform-specific catalytic differences were observed. First, the R3A isoform was found to have a significantly higher Kact for calmodulin than the other isoforms. This indicates that the variable region, which is located distal to the calmodulin-binding domain, may play a role in activation of the enzyme by calmodulin. Decreased sensitivity to calmodulin may be biologically important if free calmodulin is limiting within the neuron. The second catalytic difference noted was that the R6 isoform had a significantly lower K(m) for the peptide substrate used in this study. Although the variable region is not in the catalytic part of the enzyme, it may have an indirect function in substrate selectivity.

Structural domain mapping and phosphorylation of human erythrocyte pallidin (band 4.2).

Pallidin (band 4.2) is a major protein of the human erythrocyte membrane, and plays an important but as yet undefined role in maintaining the normal shape and lifespan of the erythrocyte. The pallidin protein has been purified by a new procedure which yields a protein which is > 97% pure as judged by gel electrophoresis, while pallidin purified by our original procedure is only approx. 85% pure. The new form of the protein is unstable in physiological salt solutions. However, taking advantage of its high purity, we have used the new form of the protein to produce a structural domain map of its principal tryptic fragments. We also show that pallidin can be phosphorylated by a red-cell membrane kinase which partially co-purifies with it, and has properties similar to the catalytic subunit of cAMP-dependent kinase. Both cAMP-dependent kinase and the red-cell kinase phosphorylate the same tryptic domains on the pallidin protein. Our results show that endogenous pallidin on the red-cell membrane is a poor substrate for the kinase, possibly because it is fully phosphorylated, or inaccessible to the kinase.

Maturation of murine erythroleukemia cells committed to differentiation requires protein kinase C.

Treatment of murine erythroleukemia cells (MELC) attached to fibronectin-coated dishes with dimethyl sulfoxide causes the cells to become committed to the erythroid differentiation pathway. These cells mature extensively and acquire the characteristics of erythroid cells. The cells lose their cell-surface fibronectin receptors and accumulate red cell-specific membrane proteins, such as band 3, in amounts comparable to those in erythrocytes. Previous studies of MELC have shown that the presence of protein kinase C (PKC) is required for commitment to differentiation, but that the level of PKC activity declines progressively during maturation. In this study, we have established a role for PKC in the maturation of MELC committed to differentiation. Our results show that down-regulation of PKC by addition of phorbol 12-myristate 13-acetate (PMA) to committed MELC blocks subsequent maturation of the cells. Treatment of MELC with the PKC inhibitors H7 and sphingosine had similar effects. Down-regulation of PKC was assayed by measuring cytosolic PKC activity as well as by Western blotting using PKC antibodies. MELC maturation was monitored by loss of the cell-surface fibronectin receptor, release of cells from fibronectin plates, and accumulation of the band 3 anion transport protein. Immunoprecipitation of surface-labeled proteins by an anti-fibronectin receptor (integrin) antibody showed that PMA-treated cultures had more fibronectin receptor protein than untreated cultures 6 days post-induction. As a result, cultures of committed MELC treated with PMA remained attached to fibronectin-coated plates, whereas non-PMA-treated cells were released into the culture medium. Furthermore, PKC-depleted cells accumulated much smaller amounts of band 3 protein and band 3 mRNA than did non-PKC-depleted controls. Our results show that although PKC activity declines progressively during post-commitment maturation of MELC, its continued presence is critical for the process of cellular maturation.

Inhibitory effect of glucose and adenosine 3',5'-monophosphate on the synthesis of inducible N-acetylglucosamine catabolic enzymes in yeast.

Glucose can block the utilization of N-acetylglucosamine in Saccharomyces cerevisiae, a facultative aerobe, but not in Candida albicans, an obligatory aerobe. Furthermore, glucose represses the synthesis of the enzymes of the N-acetylglucosamine catabolic pathway in S. cerevisiae, but not in C. albicans. The results suggest that catabolite repression is present in S. cerevisiae, but not in C. albicans. Cyclic AMP added to S. cerevisiae cells maintained in a glucose medium cannot bring about their release from catabolite repression. On the contrary, the synthesis of inducible enzymes of N-acetylglucosamine pathway was inhibited by cyclic AMP in both the yeasts. This seems to indicate that cyclic AMP can penetrate into the yeast cells. Furthermore, cyclic AMP inhibits protein synthesis, suggesting that protein synthesis in yeast is under cyclic AMP control.