Dealing with energy demand: the AMP-activated protein kinase.

The AMP-activated protein kinase (AMPK) is a member of a metabolite-sensing protein kinase family that is found in all eukaryotes. AMPK activity is regulated by vigorous exercise, nutrient starvation and ischemia/hypoxia, and modulates many aspects of mammalian cell metabolism. The AMPK yeast homolog, Snf1p, plays a major role in adaption to glucose deprivation. In mammals, AMPK also has diverse roles that extend from energy metabolism through to transcriptional control.

The Akt kinase signals directly to endothelial nitric oxide synthase.

Endothelial nitric oxide synthase (eNOS) is an important modulator of angiogenesis and vascular tone [1]. It is stimulated by treatment of endothelial cells in a phosphatidylinositol 3-kinase (PI 3-kinase)-dependent fashion by insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF) [2] [3] and is activated by phosphorylation at Ser1177 in the sequence RIRTQS(1177)F (in the single-letter amino acid code) [4]. The protein kinase Akt is an important downstream target of PI 3-kinase [5] [6], regulating VEGF-stimulated endothelial cell survival [7]. Akt phosphorylates substrates within a defined motif [8], which is present in the sequence surrounding Ser1177 in eNOS. Both Akt [5] [6] and eNOS [9] are localized to, and activated at, the plasma membrane. We found that purified Akt phosphorylated cardiac eNOS at Ser1177, resulting in activation of eNOS. Phosphorylation at this site was stimulated by treatment of bovine aortic endothelial cells (BAECs) with VEGF or IGF-1, and Akt was activated in parallel. Preincubation with wortmannin, an inhibitor of Akt signalling, reduced VEGF- or IGF-1-induced Akt activity and eNOS phosphorylation. Akt was detected in immunoprecipitates of eNOS from BAECs, and eNOS in immunoprecipitates of Akt, indicating that the two enzymes associate in vivo. It is thus apparent that Akt directly activates eNOS in endothelial cells. These results strongly suggest that Akt has an important role in the regulation of normal angiogenesis and raise the possibility that the enhanced activity of this kinase that occurs in carcinomas may contribute to tumor vascularization and survival.

Purification of calcitonin-like peptides from rat brain and pituitary.

Accumulating evidence supports the existence of nonthyroidal calcitonin (CT)-like peptides, more similar to fish CTs, which may act as endogenous regulators of CT receptors in brain and other tissues. In this study, we have carried out large-scale extractions from Sprague-Dawley rat brain diencephalon and pituitary, and purified a novel, biologically active, CT-like peptide from pituitary. Monitoring of the calcitonin-like activity of the peptides from rat brain and pituitary required different detection systems. While the brain CT cross-reacted with C-terminally directed salmon CT-specific antisera, the pituitary CT did not. However, the pituitary CT was biologically active, exhibiting specific interaction with CT receptors to activate adenylate cyclase. Conventional chromatographic techniques were employed to purify the CT-like peptides. Although the brain CT was not purified to homogeneity, size exclusion chromatography revealed the presence of multiple molecular weight forms of immunoreactive CT. Of these, only the lowest molecular weight form was biologically active. Purification from the pituitary resulted in the isolation of a biologically active peptide with a mass of 3267 Da. This mass differs from the mass of both salmon and thyroid-derived rat CT. Initial amino acid sequencing of the pituitary CT indicated that it was N-terminally blocked. Following aminopeptidase digestion, a unique six amino acid sequence, EKSQSP, was identified. Elucidation of the amino acid composition provided supporting evidence that the peptide was novel and was consistent with a full length peptide of approximately 30 amino acids. These data support the existence of novel, nonthyroidal, CTs which are potential regulators of CT receptor-mediated functions.

FHA domain boundaries of the dun1p and rad53p cell cycle checkpoint kinases.

Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues). In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks. This indicates that the non-catalytic FHA domain plays an important role in the transcriptional function of the Dun1p protein kinase.

Expression of the AMP-activated protein kinase beta1 and beta2 subunits in skeletal muscle.

A heterotrimeric member of the AMP-activated protein kinase (AMPK) isoenzyme family was purified from rat skeletal muscle by immunoaffinity chromatography, consisting of an alpha2 catalytic and two non-catalytic subunits, beta2 and gamma1. The AMPK beta2 cDNA (271 amino acids (aa), molecular weight (MW)=30¿ omitted¿307, pI 6. 3) was cloned from skeletal muscle and found to share an overall identity of 70% with beta1 (270 aa, MW=30¿ omitted¿475, pI 6.0). In the liver AMPK beta1 subunit, Ser-182 is constitutively phosphorylated whereas in skeletal muscle beta2 isoform, we find that Ser-182 is only partially phosphorylated. In addition, the autophosphorylation sites Ser-24, Ser-25 found in the beta1 are replaced by Ala-Glu in the beta2 isoform. beta2 contains seven more Ser and one less Thr residues than beta1, raising the possibility of differential post-translational regulation. Immunoblot analysis further revealed that soleus muscle (slow twitch) contains exclusively beta1 associated with alpha2, whereas extensor digitorum longus muscle alpha2 (EDL, fast twitch) associates with beta2 as well as beta1. Sequence analysis revealed that glycogen synthase, a known AMPK substrate, co-immunoprecipitated with the AMPK alpha2beta2gamma1 complex.

AMP-activated protein kinase isoenzyme family: subunit structure and chromosomal location.

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and non-catalytic, beta and gamma (38 kDa) subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and we find that rat liver AMPK-alpha2 isoform catalytic subunit is associated with beta1 and gamma1 and not with beta2 or gamma2 subunit isoforms. The beta1 and gamma1 isoforms are also subunits of the alpha1 isoform. The sequence of cloned human AMPK-beta1 is 95% identical in amino acid sequence with rat beta1. Human chromosomal localizations were determined for AMPK-alpha1 (5p11-p14), AMPK-beta1 (12q24.1-24.3) and AMPK-gamma1 (12q12-q14), respectively.

AMP-activated protein kinase phosphorylation of endothelial NO synthase.

The AMP-activated protein kinase (AMPK) in rat skeletal and cardiac muscle is activated by vigorous exercise and ischaemic stress. Under these conditions AMPK phosphorylates and inhibits acetyl-coenzyme A carboxylase causing increased oxidation of fatty acids. Here we show that AMPK co-immunoprecipitates with cardiac endothelial NO synthase (eNOS) and phosphorylates Ser-1177 in the presence of Ca2+-calmodulin (CaM) to activate eNOS both in vitro and during ischaemia in rat hearts. In the absence of Ca2+-calmodulin, AMPK also phosphorylates eNOS at Thr-495 in the CaM-binding sequence, resulting in inhibition of eNOS activity but Thr-495 phosphorylation is unchanged during ischaemia. Phosphorylation of eNOS by the AMPK in endothelial cells and myocytes provides a further regulatory link between metabolic stress and cardiovascular function.

Simplified conjugation chemistry for coupling peptides to F(ab') fragments: autologous red cell agglutination assay for HIV-1 antibodies.

The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on agglutination of autologous red blood cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579-613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous red cell agglutination test is described. F(ab')2 fragments of the anti-glycophorin antibody were prepared by pepsin digestion and reduced to F(ab') fragments with the use of tri-n-butylphosphine (TBP). This permitted the simultaneous reduction of the F(ab') fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579-613) [Cys-Acm 598, Lys-BrAc 604] containing epsilon-bromoacetyl-lysine at residue 604 to the resultant F(ab') fragment. Conjugation to the F(ab') fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab'). The resultant F(ab')-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous red cell agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

Metorphamide, a novel endogenous adrenal opioid peptide, inhibits nicotine-induced secretion from bovine adrenal chromaffin cells.

Opioid peptides are found in high concentrations in the adrenal medulla. Recently, a novel opioid octapeptide, metorphamide, possessing an amidated C-terminal, was characterized and also found to be present in adrenal tissue. We have studied the ability of this novel peptide to modify nicotine-induced secretion from isolated bovine adrenal chromaffin cells. Exocytosis was monitored by measuring adenosine triphosphate (ATP) release on-line by the luciferin-luciferase bioluminescence method, or by measuring endogenous catecholamine release by high-performance liquid chromatography (HPLC) with electrochemical detection. Metorphamide inhibited 5 microM nicotine-induced ATP release from fresh chromaffin cells by almost 50% at 5 microM. Metorphamide at concentrations less than 1 microM had no effect on 5 microM nicotine-induced adrenaline and noradrenaline release from cultured cells, but at higher concentrations inhibited their release equally, with an IC50 of approximately 10 microM. By contrast, Met5-enkephalin inhibited the release of both catecholamines equally with an IC50 of greater than 1 mM, making metorphamide greater than 100-fold more potent than Met5-enkephalin in this system. Naloxone (10 microM) and diprenorphine (1 microM) failed to antagonise the inhibitory action of metorphamide on nicotine-induced catecholamine release. Metorphamide inhibited the nicotinic response in a non-competitive manner, and failed to affect either adrenaline or noradrenaline release induced by elevated potassium ion concentrations. The results suggest metorphamide acts on naloxone- and diprenorphine-resistant receptors to inhibit chromaffin cell nicotinic secretion and that the novel amidated C-terminal of the peptide is important for this action.

Autologous red cell agglutination test for antibodies to feline immunodeficiency virus.

The T-lymphotropic lentivirus, feline immunodeficiency virus (FIV) is now recognised as a major viral pathogen affecting domestic cat populations worldwide. A rapid, autologous red cell agglutination test for antibodies to FIV has been developed. A synthetic peptide analog corresponding to the immunodominant epitope within the FIV transmembrane glycoprotein gp40 residues (680-715) KVEAMEKFLYTAFAMQELGC (Acm)NQNQFFK(BrAc)KIPLELWTR was conjugated to an anti-feline erythrocyte antibody using a thio-ether linkage. Within 3 min of adding this reagent to 20 microliters of whole blood, circulating antibody to the peptide epitope caused agglutination of the red blood cells. The performance of this simple test is comparable with the two commercially available enzyme immunoassay (EIA) kits and an EIA based on this peptide. A variant of the gp40 (680-715) peptide corresponding to the FIV, PPR strain gp40 (678-716) sequence was also synthesised and no difference in reactivity was observed in an EIA on 211 seropositive samples, indicating that the peptide-based test may be applicable to other known strains of the virus.

Effects of opioid peptides containing the sequence of Met5-enkephalin or Leu5-enkephalin on nicotine-induced secretion from bovine adrenal chromaffin cells.

Eighteen endogenous opioid peptides, all containing the sequence of either Met5- or Leu5-enkephalin, were tested for their ability to modify nicotine-induced secretion from bovine adrenal chromaffin cells. ATP released from suspensions of freshly isolated cells was measured with the luciferin-luciferase bioluminescence method as an index of secretion. None of the peptides affected 5 microM nicotine-induced ATP release at 10 nM. Three peptides inhibited secretion at 5 microM: dynorphin1-13, dynorphin1-9, and rimorphin inhibited by 65%, 37%, and 29% respectively. Use of peptidase inhibitors (bestatin, thiorphan, bacitracin, or 1,10-phenanthroline) did not result in any of the other peptides showing potent actions on the nicotinic response, although bestatin and thiorphan did enhance the inhibitory actions of dynorphin1-13 and dynorphin1-9 by 20-30%. Nicotine-induced secretion of endogenous catecholamines from bovine chromaffin cells cultured for 3 days was also studied to assess any selective actions of the peptides on adrenaline or noradrenaline cell types. Dynorphin1-13 was 1,000-fold more potent than Leu5-enkephalin at inhibiting endogenous catecholamine secretion. Dynorphin1-13 was slightly more potent at inhibiting noradrenaline release than adrenaline release whereas Leu5-enkephalin showed the opposite selectivity. The structure-activity relationships of opioid peptide actions on the chromaffin cell nicotinic response are discussed in relation to the properties of the adrenal opioid binding sites.

Role of the pseudosubstrate sequence in smooth muscle myosin light chain kinase thermal stability.

Smooth muscle myosin light chain kinase (MLCK) is stable in the presence of Ca2+/calmodulin and does not undergo inactivation as reported for skeletal muscle MLCK (Kennelly, P.J., Starovasnik, M.A., Edelman, A.M., and Krebs, E.G. (1990) J. Biol. Chem. 265, 1742-1749). The 61-kDa tryptic fragment of smMLCK-(283-779) with the pseudosubstrate/calmodulin binding sequence deleted undergoes rapid inactivation (t1/2 = 5 min at 25 degrees C). Thermal inactivation renders the 61-kDa fragment more susceptible to cleavage by trypsin. The pseudosubstrate sequence, smMLCK-(787-807) prevents inactivation with high potency (half-maximal protective concentration, PC0.5 = 102 +/- 9 nM). The hexapeptide smMLCK-(797-802), Arg-Arg-Lys-Trp800-Gln-Lys, protected with a similar potency (PC0.5 = 73 +/- 14 nM). The four basic residues as well as Trp were important for maintaining protection by the hexapeptide smMLCK-(797-802). Substitution of Trp800 with Ala or Leu increased the PC0.5 to 500 nM. However, substitution of both aromatic residues Tyr794 and Trp800 in the longer pseudosubstrate peptide-(787-807) had little effect, indicating that with the longer peptide other multiple interactions were sufficient to stabilize the enzyme. The peptide substrate MLC-(11-23) A14,15 was also protective (PC0.5 = 380 nM) as was Mg(2+)-ATP, Mg(2+)-ADP, and Mg2+ plus adenosine. The results demonstrate that the sequence extending from 787-815 encoding the previously identified overlapping pseudosubstrate and calmodulin binding sequences also contains residues that are essential for maintaining thermal stability but these exhibit distinct structure/function relationships.

Plasma amino-terminal pro-brain natriuretic peptide: a novel approach to the diagnosis of cardiac dysfunction.

BACKGROUND: Heart failure is a common cause of hospitalization and death across the industrialized world. Improving the diagnosis and care of patients with heart failure is therefore likely to have a major impact on morbidity, mortality, and health care costs. METHODS AND RESULTS: To determine the relation between cardiac function and plasma levels of amino-terminal brain natriuretic peptide precursor (NT-proBNP), plasma NT-proBNP levels and ventricular function (by radionucleotide ventriculography) were measured in healthy patients, patients with renal failure, patients with recent myocardial infarction, and patients investigated for cardiorespiratory symptoms. Plasma NT-proBNP levels were greater in healthy women (median, 1.5 fmol/mL; range, 1.0 to 13.8 fmol/mL; n = 34) than healthy men (median, 1.0 fmol/mL; range, 1.0 to 3.3 fmol/mL; n = 33; P = .012). NT-proBNP levels were elevated in subjects with renal failure (geometric mean, 314 fmol/mL; range, 18 to 5,800 fmol/mL) and were related to left ventricular ejection fraction (LVEF) (r = -0.86; P < .0001; n = 19). NT-proBNP levels were also related to LVEF in patients with recent myocardial infarction (r = -0.62; P = .0003; n = 29) and those investigated for cardiorespiratory symptoms (r = -0.56; P < .0001; n = 129). Applying an upper limit of normal of 5 fmol/mL for men and 15 fmol/mL for women (specificity, 100%), elevated plasma NT-proBNP levels had 100% sensitivity for the detection of LVEF less than 45% after myocardial infarction and 97% sensitivity for the detection of LVEF less than 45% in patients investigated for cardiorespiratory symptoms. NT-proBNP levels were also elevated in 87% of the patients with normal systolic function (LVEF > or = 45%) after myocardial infarction and in 87% of the patients investigated for cardiorespiratory symptoms with heart failure and normal systolic function (LVEF > or = 45%). CONCLUSIONS: Plasma NT-proBNP level is a sensitive indicator of cardiac dysfunction, both in the presence and absence of systolic dysfunction, and may prove to be a useful tool for the identification and management of cardiac dysfunction in the general community.

Chicken smooth muscle myosin light chain kinase is acetylated on its NH2-terminal methionine.

The reported cDNA structure of chicken smooth muscle myosin light chain kinase (smMLCK) encodes a protein of 972 residues (Olson et al. Proc. Natl. Acad. Sci USA, 87:2284-2288, 1990). The calculated M(r) is 107,534 whereas the estimate by SDS-PAGE is approximately 130,000. Gibson and Higgins (DNA Sequence (in press)) have recently reported the possibility of errors in the cDNA sequence for non-muscle MLCK and that the NH2-terminus of both it and smMLCK may extend beyond the reported coding region. The native smMLCK is NH2-terminally blocked. A CNBr peptide derived from smMLCK contains the NH2-terminal sequence Asp-Phe-Arg-Ala corresponding to residues 2 to 4 in the smMLCK sequence indicating that Met-1 is present. Using a limited thermolysin digest we isolated an NH2-terminally blocked peptide by reversed-phase HPLC. This thermolytic peptide had a mass of approximately 797 by time of flight mass spectrometry. Amino acid analysis and Edman sequencing of a CNBr-subfragment of the thermolytic peptide indicated that it had the composition and sequence, (Met)-Asp-Phe-Arg-Ala-Asn, with a calculated mass of 753. The difference in mass corresponds to the NH2-terminal Met being blocked by acetylation. The results demonstrate that the NH2-terminal sequence of smMLCK inferred from the reported cDNA sequence is correct and that the proposed initiating Met is not removed, but modified by alpha-NH2 acetylation of the translation product.

Myosin light chain kinase autoregulatory pseudosubstrate prototope.

The myosin light chain kinase is catalytically inactive unless activated by calmodulin. An autoregulatory pseudosubstrate region located on the carboxyl-terminal side of the enzyme's catalytic domain is responsible for maintaining the enzyme in a latent form. This pseudosubstrate region overlaps the calmodulin binding domain. Synthetic peptides corresponding to the regulatory region can have both substrate antagonist and calmodulin antagonist activities. The pseudosubstrate peptide from the smooth muscle myosin light chain kinase, smMLCK(787-807), S787KDRMKKYMARRKW800QKTGHAV807 is a potent substrate antagonist with a Ki of approximately 12 nM and acts as a calmodulin antagonist with an IC50 = 0.54 microM. The shorter peptide R797RKWQK802, Ki = 1.26 microM, is the core region primarily responsible for substrate antagonist activity and is a weak calmodulin antagonist, IC50 = 181 microM. The corresponding skeletal muscle peptide, KRRWKK was a comparable substrate antagonist, IC50 = 1.63 microM, but a 30-fold more potent calmodulin antagonist, IC50 = 6.1 microM. Substitution of the Trp in either peptide with Phe or Leu did not significantly alter the substrate antagonist activity but markedly reduced calmodulin antagonist activity, RRKWQK, IC50(calmodulin) = 181 microM; RRKFQK, IC50(calmodulin) = 488 microM; RRKLQK, IC50(calmodulin) = 1700 microM; KRRWKK, IC50(calmodulin) = 6.1 microM; KRRLKK, IC50(calmodulin) = 221 microM and KRRFKK, IC50(calmodulin) = 93 microM. The IC50(substrate) values for these peptides ranged from 0.5-13 microM. The peptide KRRLKK was the most selective substrate antagonist and is suitable as an inhibitor for the myosin light chain kinase with the ratio IC50(calmodulin): IC50(substrate) = 273 and an IC50(substrate) = 0.81 microM.

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins.

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3delta977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3delta977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

Synapsins as major neuronal Ca2+/S100A1-interacting proteins.

The mammalian S100A1 protein can activate the invertebrate myosin-associated giant protein kinase twitchin in a Ca(2+)-dependent manner by more than 1000-fold in vitro; however, no mammalian S100-dependent protein kinases are known. In an attempt to identify novel mammalian Ca(2+)/S100A1-regulated protein kinases, brain extracts were subjected to combined Ca(2+)-dependent affinity chromatography with S100A1 and an ATP analogue. This resulted in the purification to near-homogeneity of the four major synapsin isoforms Ia, Ib, IIa and IIb. All four synapsins were specifically affinity-labelled with the ATP analogue 5'-p-fluorosulphonylbenzoyladenosine. S100A1 bound to immobilized synapsin IIa in BIAcore experiments in a Ca(2+)-dependent and Zn(2+)-enhanced manner with submicromolar affinity; this interaction could be competed for with synthetic peptides of the proposed S100A1-binding sites of synapsin. Double-labelling confocal immunofluorescence microscopy demonstrated that synapsins and S100A1 are both present in the soma and neurites of PC12 cells, indicating their potential to interact in neurons in vivo.

Mammalian AMP-activated protein kinase shares structural and functional homology with the catalytic domain of yeast Snf1 protein kinase.

The AMP-activated protein kinase is responsible for the regulation of fatty acid synthesis by phosphorylation of acetyl-CoA carboxylase. It may also regulate cholesterol synthesis via phosphorylation and inactivation of hormone-sensitive lipase and hydroxymethylglutaryl-CoA reductase. We have purified the AMP-activated protein kinase 14,000-fold from porcine liver. The 63-kDa catalytic subunit co-purifies with two proteins of 40 and 38 kDa that may function as subunits. Partial amino acid sequence of the 63-kDa subunit revealed a striking homology with the catalytic domain of the yeast protein kinase transcriptional regulator Snf1 and its plant homologs. The Snf1 (72 kDa) and Snf4 (36 kDa) complex was also purified and found to phosphorylate the AMP-activated protein kinase peptide substrate, HMRSAMSGLHLVKRR-amide, but was not activated by AMP. Both Snf1/4 and the AMP-activated protein kinase phosphorylate and inactivate yeast acetyl-CoA carboxylase in vitro. These results indicate that during evolution the catalytic domain sequences of the Snf1 protein kinase subfamily have been exploited in the control of mammalian lipid metabolism and raise the possibilities that the AMP-activated protein kinase may have other substrates involved in regulating gene expression pathways, as well as Snf1 homologs participating in the control of lipid metabolism in many eukaryotic organisms.

Human factor H-related protein 5 (FHR-5). A new complement-associated protein.

A novel human plasma protein has been identified as a universal component of complement deposits, when complement is detected immunohistochemically in vivo. The protein is homologous to complement factor H and related proteins and has been designated factor H-related protein 5 (FHR-5). FHR-5 was identified by a monoclonal antibody raised using pathologic human glomerular preparations as the immunogen. FHR-5 was purified by affinity chromatography from complement-lysed erythrocytes, and the peptide sequence was obtained. The cDNA was cloned from a human liver library, and FHR-5 was deduced to be a protein containing 551 amino acids organized into nine short consensus repeat motifs. The short consensus repeats of FHR-5 show homology to Factor H and to other Factor H-related proteins, with some unique features demonstrated. Recombinant FHR-5, expressed in insect cells, was shown to bind C3b in vitro. The strong association of FHR-5 with tissue complement deposits in vivo suggests that this additional member of the Factor H family of proteins has a function in complement regulation.

Posttranslational modifications of the 5'-AMP-activated protein kinase beta1 subunit.

The AMP-activated protein kinase (AMPK) consists of catalytic alpha and noncatalytic beta and gamma subunits and is responsible for acting as a metabolic sensor for AMP levels. There are multiple genes for each subunit and the rat liver AMPK alpha1 and alpha2 catalytic subunits are associated with beta1 and gamma1 noncatalytic subunits. We find that the isolated gamma1 subunit is N-terminally acetylated with no other posttranslational modification. The isolated beta1 subunit is N-terminally myristoylated. Transfection of COS cells with AMPK subunit cDNAs containing a nonmyristoylatable beta1 reduces, but does not eliminate, membrane binding of AMPK heterotrimer. The isolated beta1 subunit is partially phosphorylated at three sites, Ser24/25, Ser182, and Ser108. The Ser24/25 and Ser108 sites are substoichiometrically phosphorylated and can be autophosphorylated in vitro. The Ser-Pro site in the sequence LSSS182PPGP is stoichiometrically phosphorylated, and no additional phosphate is incorporated into this site with autophosphorylation. Based on labeling studies in transfected cells, we conclude that alpha1 Thr172 is a major, although not exclusive, site of both basal and stimulated alpha1 phosphorylation by an upstream AMPK kinase.