Studies have suggested that the expression, translocation, and function of a4b2 nicotinic receptors may be modulated by a4 subunit phosphorylation, but little direct evidence exists to support this idea. The objective of these experiments was to identify specific serine/threonine residues on a4 subunits that are phosphorylated in vivo by cAMP-dependent protein kinase and protein kinase C (PKC). To accomplish this, DNAs coding for human a4 subunits containing alanines in place of serines/threonines predicted to represent phosphorylation sites were constructed, and transiently transfected with the DNA coding for wild-type b2 subunits into SH-EP1 cells. Cells were pre-incubated with 32 Pi and incubated in the absence or presence of forskolin or phorbol 12,13-dibutyrate. Immunoprecipitated a4 subunits were subjected to immunoblot, autoradiographic and phosphoamino acid analyses, and two-dimensional phosphopeptide mapping. Results confirmed the presence of two a4 protein bands, a major band of 71/75 kDa and a minor band of 80/ 85 kDa. Phosphoamino acid analysis of the major band indicated that only serine residues were phosphorylated. Phosphopeptide maps demonstrated that Ser362 and 467 on the M3/M4 cytoplasmic domain of the a4 subunit represent major cAMP-dependent protein kinase phosphorylation sites, while Ser550 also contained within this major intracellular loop is a major site for protein kinase C phosphorylation.

Rhodobacter sphaeroides f. sp. denitrificans biotin sulfoxide reductase has been heterologously expressed in Escherichia coli as a functional 106-kDa glutathione S-transferase fusion protein. Following cleavage with Factor Xa and purification to homogeneity, the soluble 83-kDa enzyme retained biotin sulfoxide reductase activity using reduced methyl viologen or reduced benzyl viologen as artificial electron donors. Initial rate kinetics indicated a specific activity at pH 8.0 of 0.9 mol of biotin sulfoxide reduced per min/nmol of enzyme and K m values of 29 and 15 M for reduced methyl viologen and biotin sulfoxide reductase, respectively. Biotin sulfoxide reductase was also capable of reducing nicotinamide N-oxide, methionine sulfoxide, trimethylamine-N-oxide, and dimethyl sulfoxide, although with varying efficiencies, and could directly utilize NADPH as a reducing agent, both for the reduction of biotin sulfoxide and ferricyanide. The enzyme contained the prosthetic group, molybdopterin guanine dinucleotide, and did not require any accessory proteins for functionality. These results represent the first successful heterologous expression and characterization of a functional molybdopterin guanine dinucleotide-containing enzyme and the demonstration of reduced pyridine nucleotide-dependent biotin sulfoxide reductase activity.Biotin sulfoxide (BSO) 1 reductase, which catalyzes the reduction of d-biotin d-sulfoxide to d-biotin according to the following scheme,has been postulated to function in a variety of roles in bacterial metabolism (Pierson and Campbell, 1990) including scavenging biotin sulfoxide from the environment and thus allowing bacteria to utilize this oxidized form of biotin for biosynthetic reactions: reducing bound intracellular biotin, such as bound to biotin-containing carboxylases or protein degradation products, that have become oxidized in aerobic environments; or as a potential protector of the cell from oxidative damage similar to the proposed roles of methionine sulfoxide reductase (Ejiri et al., 1980;Brot et al., 1981;Rahman et al., 1992) and superoxide dismutase (Fridovich, 1989).BSO reductase has been partially purified from Escherichia coli and demonstrated to be a soluble protein that requires an unidentified form of the Mo cofactor (Rajagopalan and Johnson, 1992) and several accessory proteins for activity (del CampilloCampbell and Campbell, 1982). These accessory proteins include a small, heat stable, thioredoxin-like protein moiety, referred to as protein-(SH) 2 and which functions as a source of reducing equivalents and an unidentified flavoprotein (del Campillo-Campbell et al., 1979). The extensive characterization of BSO reductase has been limited by the low natural abundance of the protein, its constitutive expression, and the requirement for auxiliary proteins for activity coupled with the difficulty of its detection in the absence of specific antibodies. While the E. coli BSO reductase bisC structural gene has been cloned and sequenced, the enzyme has not been produced using hete...

Neuronal nicotinic receptor α4 subunits associated with nicotinic α4β2 receptors are phosphorylated by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), but the stages of receptor formation during which phosphorylation occurs and the functional consequences of kinase activation are unknown. SH-EP1 cells transfected with DNAs coding for human α4 and/or β2 subunits were incubated with 32 Pi, and PKA or PKC were activated by forskolin or phorbol 12,13-dibutyrate, respectively. Immunoprecipitation and immunoblotting of proteins from cells expressing α4β2 receptors or only α4 subunits were used to identify free α4 subunits, and α4 subunits present in immature α4β2 complexes and mature α4β2 pentamers containing complex carbohydrates. In the absence of kinase activation, phosphorylation of α4 subunits associated with mature pentamers was 3 times higher than subunits associated with immature complexes. PKA and PKC activation increased phosphorylation of free α4 subunits on different serine residues; only PKC activation phosphorylated subunits associated with mature α4β2 receptors. Activation of both PKA and PKC increased the density of membrane-associated receptors, but only PKC activation increased peak membrane currents. PKA and PKC activation also phosphorylated β2 subunits associated with mature α4β2 receptors. Results indicate that activation of PKA and PKC leads to the phosphorylation α4β2 receptors at different stages of receptor formation and maturation and has differential effects on the expression and function of α4β2 receptors. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptNeuroscience. Author manuscript; available in PMC 2010 February 18. Published in final edited form as:Neuroscience. (Zoli et al., 1995). These α4β2 receptors participate in numerous biochemical and physiological processes, have been implicated in several neurological and behavioral disorders including nocturnal frontal lobe epilepsy and Alzheimer's disease, and may be responsible for the rewarding and addictive effects of nicotine (Picciotto et al., 2001;Tapper et al., 2004).Studies have suggested that the expression and function of α4β2 receptors are regulated posttranslationally through phosphorylation/dephosphorylation mechanisms involving both cAMP-dependent protein kinase (PKA) and protein kinase C (PKC) (Rothhut et al., 1996;Eilers et al., 1997;Gopalakrishnan et al., 1997;Fenster et al., 1999;Jeanclos et al., 2001;Nashmi et al., 2003;Exley et al., 2006). Initial studies using M10 fibroblasts stably transfected with chicken α4 and β2 su...