Tyrosine hydroxylase activity is regulated by two distinct dopamine-binding sites

Tyrosine hydroxylase (TH), the rate-limiting enzyme in the biosynthesis of the catecholamines dopamine, noradrenaline and adrenaline, is regulated acutely by feedback inhibition by the catecholamines and relief of this inhibition by phosphorylation of serine 40 (Ser40). Phosphorylation of serine 40 abolishes the binding of dopamine to a high affinity ( K _D < 4 nM) site on TH, thereby increasing the activity of the enzyme. We have found that TH also contains a second low affinity ( K _D = 90 nM) dopamine-binding site, which is present in both the non-phosphorylated and the Ser40-phosphorylated forms of the enzyme. Binding of dopamine to the high-affinity site decreases V _max and increases the K _m for the cofactor tetrahydrobiopterin, while binding of dopamine to the low-affinity site regulates TH activity by increasing the K _m for tetrahydrobiopterin. Kinetic analysis indicates that both sites are present in each of the four human TH isoforms. Dissociation of dopamine from the low-affinity site increases TH activity 12-fold for the non-phosphorylated enzyme and 9-fold for the Ser40-phosphorylated enzyme. The low-affinity dopamine-binding site has the potential to be the primary mechanism responsible for the regulation of catecholamine synthesis under most conditions.     

nifH promoter activity is regulated by DNA supercoiling in Sinorhizobium meliloti

In prokaryotes, DNA supercoiling regulates the expression of many genes; for example, the expression of Klebsiella pneumoniae nifLA operon depends on DNA negative supercoiling in anaerobically grown ceils, which indicates that DNA supercoiling might play a role in gene regulation of the anaerobic response. Since the expression of the nifH promoter in Sinorhizobium meliloti is not repressed by oxygen, it is proposed that the status of DNA supercoiling may not affect the expression of the nifH promoter. We tested this hypothesis by analyzing nifH promoter activity in wild-type and gyr- Escherichia coli in the presence and absence of DNA gyrase inhibitors. Our results show that gene expression driven by the S.meliloti nifH promoter requires the presence of active DNA gyrase. Because DNA gyrase increases the number of negative superhelical turns in DNA in the presence of ATP, our data indicate that negative supercoiling is also important for nifH promoter activity. Our study also shows that the DNA supercoiling-dependent S. meliloti nifH promoter activity is related to the trans-acting factors NtrC and NifA that activate it. DNA supercoiling appeared to have a stronger effect on NtrC-activated nifH promoter activity than on NifA-activated promoter activity. Collectively, these results from the S. meliloti nifH promoter model system seem to indicate that, in addition to regulating gene expression during anaerobic signaling, DNA supercoiling may also provide a favorable topology for trans-acting factor binding and promoter activation regardless of oxygen status.     

Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2

Glycosylphosphatidylinositols (GPIs) are attached to the C-termini of many proteins, thereby acting as membrane anchors. Biosynthesis of GPI is initiated by GPI-N-acetylglucosaminyltransferase (GPI-GnT), which transfers N-acetylglucosamine from UDP- N-acetylglucosamine to phosphatidylinositol. GPI-GnT is a uniquely complex glycosyltransferase, consisting of at least four proteins, PIG-A, PIG-H, PIG-C and GPI1. Here, we report that GPI-GnT requires another component, termed PIG-P, and that DPM2, which regulates dolichol-phosphate-mannose synthase, also regulates GPI-GnT. PIG-P, a 134-amino acid protein having two hydrophobic domains, associates with PIG-A and GPI1. PIG-P is essential for GPI-GnT since a cell lacking PIG-P is GPI-anchor negative. DPM2, but not two other components of dolichol-phosphate-mannose synthase, associates with GPI-GnT through interactions with PIG-A, PIG-C and GPI1. Lec15 cell, a null mutant of DPM2, synthesizes early GPI intermediates, indicating that DPM2 is not essential for GPI-GnT; however, the enzyme activity is enhanced 3-fold in the presence of DPM2. These results reveal new essential and regulatory components of GPI-GnT and imply co-regulation of GPI-GnT and the dolichol-phosphate-mannose synthase that generates a mannosyl donor for GPI.     

RNase activity requires formation of disulfide bonds and is regulated by the redox state

The activity of many RNases requires the formation of one or more disulfide bonds which can contribute to their stability. In this study, we show that RNase activity and, to a much lesser extent, nuclease activity, are redox regulated. Intracellular RNase activity was altered in vitroby changes in the glutathione redox state. Moreover, RNase activity was abolished following exposure to reducing agents such as -ME or DTT. Following reduction with glutathione (GSH), RNase activity could be fully reactivated with oxidized glutathione (GSSG). In contrast, RNase activity could not be reactivated when reduced with DTT. Decreasing the level of glutathione in vivoin wheat increased RNase activity. Tobacco engineered to have an increased glutathione redox state exhibited substantially lower RNase activity during dark-induced senescence. These results suggest that RNase activity requires the presence of one or more disulfide bonds that are regulated by glutathione and demonstrate for the first time that RNase activity can be altered with an alteration in cellular redox state.     

The functioning of the Drosophila CPEB protein Orb is regulated by phosphorylation and requires casein kinase 2 activity

The Orb CPEB protein regulates translation of localized mRNAs in Drosophila ovaries. While there are multiple hypo- and hyperphosphorylated Orb isoforms in wild type ovaries, most are missing in orb(F303), which has an amino acid substitution in a buried region of the second RRM domain. Using a proteomics approach we identified a candidate Orb kinase, Casein Kinase 2 (CK2). In addition to being associated with Orb in vivo, we show that ck2 is required for orb functioning in gurken signaling and in the autoregulation of orb mRNA localization and translation. Supporting a role for ck2 in Orb phosphorylation, we find that the phosphorylation pattern is altered when ck2 activity is partially compromised. Finally, we show that the Orb hypophosphorylated isoforms are in slowly sedimenting complexes that contain the translational repressor Bruno, while the hyperphosphorylated isoforms assemble into large complexes that co-sediment with polysomes and contain the Wisp poly(A) polymerase.