Recombinantly expressed human ATP:citrate lyase was purified from E. coli, and its kinetic behavior was characterized before and after phosphorylation. Cyclic AMP-dependent protein kinase catalyzed the incorporation of only 1 mol of phosphate per mole of enzyme homotetramer, and glycogen synthase kinase-3 incorporated an additional 2 mol of phosphate into the phosphorylated protein. Isoelectric focusing revealed that all of the phosphates were incorporated into only one of the four enzyme subunits. Phosphorylation resulted in a 6-fold increase in V(max) and the conversion of citrate dependence from sigmoidal, displaying negative cooperativity, to hyperbolic. The phosphorylated recombinant enzyme is more similar to the enzyme isolated from mammalian tissues than unphosphorylated enzyme with respect to the K(m) for citrate, CoA, and ATP, and the specific activity. Fructose 6-phosphate was found to be a potent activator (60-fold) of the unphosphorylated recombinant enzyme, with half-maximal activation at 0.16 mM, which results in a decrease in the apparent K(m) for citrate and ATP, as well as an increase in the V(max) of the reaction. Thus, human ATP:citrate lyase activity is regulated in vitro allosterically by phosphorylated sugars as well as covalently by phosphorylation.

Abstract-The HCN family of ion channel subunits underlies the currents I f in heart and I h and I q in the nervous system.In the present study, we demonstrate that minK-related peptide 1 (MiRP1) is a ␤ subunit for the HCN family. As such, it enhances protein and current expression as well as accelerating the kinetics of activation. Because MiRP1 also functions as a ␤ subunit for the cardiac delayed rectifier I Kr , these results suggest that this peptide may have the unique role of regulating both the inward and outward channels that underlie cardiac pacemaker activity. The full text of this article is available at http://www.circresaha.org. (Circ Res. 2001;88:e84-e87.)Key Words: HCN family Ⅲ MiRP1 Ⅲ KCNE family Ⅲ ␤ subunit T he HCN (hyperpolarization-activated cyclic nucleotidegated) family of ion channel subunits has been identified as the molecular correlate of the currents I f in heart and I h and I q in neurons. [1][2][3] However, several ion channels are heteromultimers of a large ␣ subunit (like the HCN family members) and smaller ␤ subunits. The cardiac delayed rectifiers I Kr 4 and I Ks 5 are examples of this basic principle. Their ␣ subunits derive from the ERG and KCNQ families, respectively, but both also contain ␤ subunits from the KCNE family of single transmembranespanning proteins called minK and minK-related peptides (MiRPs). In this study, we report that MiRP1 enhances the expression and speeds the kinetics of activation of the HCN family of channel subunits. From immunoprecipitation experiments, we show that it most probably forms a complex with HCN1. Using RNase protection assays (RPAs), we demonstrate that MiRP1 mRNA is prevalent in the primary cardiac pacemaking region, the sinoatrial (SA) node, and barely detectable in ventricle. Cardiac pacemaker activity is generated by a narrow balance of inward (I f ) and outward (I Kr ) currents. Our results demonstrate for the first time the potential importance of a single ␤ subunit in simultaneously regulating both the expression and gating of both inward and outward cardiac pacemaker channels. Materials and Methods Heterologous Expression in Xenopus OocytescRNA encoding mouse HCN1 or HCN2, rat MiRP1 with or without an HA tag at the carboxy-terminal, and rat minK were transcribed using the mMessage mMachine kit (Ambion). Xenopus laevis oocytes were isolated, injected with 2 to 5 ng (50 to 100 nL) of cRNA, and maintained in Barth medium at 18°C for 1 to 3 days. For experiments using both HCN1 or HCN2 and MiRP1 or minK, the respective cRNAs were injected in a 1:0.04 to 1 ratio. Electrophysiological studies on oocytes used the 2-microelectrode voltage clamp. The extracellular recording solution (OR2) contained, in mmol/L, NaCl 80, KCl 2, MgCl 2 1, and Na-HEPES 5 (pH 7.6). Group data are presented as meanϮSEM. Tests of statistical significance for midpoint and slope of activation curves were performed using unpaired Student's t tests. PϽ0.05 is considered significant. RNase Protection AssaysThe procedures for the preparation of total RNA from rabbit he...

Multifunctional protein kinase (MFPK) phosphorylates ATP-citrate lyase on peptide B on two sites, BT and BS, on threonine and serine, respectively, inhibitor 2 on a threonyl residue, and glycogen synthase at sites 2 and 3. The phosphorylation sites BT and BS of ATP-citrate lyase are dependent on prior phosphorylation at site A whereas site A phosphorylation is decreased by prior phosphorylation at sites BT and BS. To study the MFPK recognition sites and the site-site interactions, the amino acid sequences of ATP-citrate lyase peptide B and inhibitor 2 were determined and compared to each other and to glycogen synthase sites 3-5. The sequence of the tryptic peptide containing the two phosphorylation sites of peptide B is -Phe-Leu-Leu-Asn-Ala-Ser-Gly-Ser-Thr-Ser-Thr(P)-Pro-Ala-Pro-Ser(P)-Arg-, and the sequence of the MFPK phosphorylation site of inhibitor 2 is -Ile-Asp-Glu-Pro-Ser-Thr(P)-Pro-Tyr-. This inhibitor 2 site is identical with the site phosphorylated by glycogen synthase kinase 3/FA. These results suggest that at least some of the sites phosphorylated by MFPK (BT of ATP-citrate lyase, Thr 72 of inhibitor 2, and sites 3b and 4 of glycogen synthase) contain a Ser/Thr flanked by a carboxyl-terminal proline. However, as MFPK did not phosphorylate a series of peptides containing the -X-Thr/Ser-Pro-X- sequence, this minimum consensus sequence is not sufficient for phosphorylation by MFPK.(ABSTRACT TRUNCATED AT 250 WORDS)

Multifunctional ATP-citrate lyase kinase (ACLK) exhibits several properties that are similar to glycogen-synthase kinase-3 (GSK-3). The molecular cloning of two distinct mammalian GSK-3 cDNAs and a Drosophila melanogaster (fruitfly) homologue, zeste-white3sgg, has established the existence of a GSK-3 subfamily. A multifunctional protein kinase first identified as an ACLK has recently been shown to exhibit several similarities to the alpha- and beta-forms of GSK-3. Here we have used immunological and biochemical analyses to directly compare these enzymes. Thus purified preparations of ACLK isolated from brain and liver preferentially cross-react with anti-GSK-3 alpha antisera and phosphorylate previously defined substrates of GSK-3 at identical sites. Conversely, both alpha- and beta-forms of GSK-3 phosphorylated ATP-citrate lyase at the same site(s) targeted by ACLK. These, and other similarities, demonstrate ACLK to be identical with, or highly related to, GSK-3 alpha, the implications of which are discussed.