Proc. Nati. Acad. Sci. USA Vol. 83, pp. 6267-6271, September 1986 Biochemistry 2'-Phosphoadenylylation of eukaryotic proteins: A type of covalent modification (ADP-ribose/phosphoADP-ribosylation/NADP labeling/microsomes/mitochondria) HELMUTH HILZ, WERNER FANICK, AND KARIN KLAPPROTH Institut fUr Physiologische Chemie der Universit~it, 2000 Hamburg 20, Federal Republic of Germany Communicated by E. R. Stadtman, May 1, 1986 ABSTRACT An enzymatic system in rat liver microsomal NH2 * preparations has been detected that catalyzes the transfer ofthe 2'-phospho-AMP moiety from NADP to endogenous poly- peptides; the major acceptor is a polypeptide of about 40 kDa (p40). Modification of the acceptor by 2'-phospho-AMP resi- //0 dues was deduced from the simultaneous transfer of 2'- [33P]phosphate and [3H~adenine residues from double-labeled C - NH2 NADP, while no incorporation of radioactivity into p40 was seen with NADP species labeled in the NMN moiety. The true N I 0~~~~~~~~~~1** substrate of this phosphoadenylylation reaction was 2'- phospho-ADP-ribose rather than NADP, because labeled HO OH phospho-ADP-ribose was as efficient as or more efficient than I HO O NADP in forming modified p40. Also, NADP was rapidly converted to phospho-ADP-ribose during incubation with 0I * microsomes. Furthermore, isonicotinic acid hydrazide, an I civ- - -- inhibitor of NADP glycohydrolase, prevented phosphoadenyl- I ~~~~~Active P-AMP ylation from NADP, but not from phospho-ADP-ribose, and glycohydrolase-resistant NADPH could not substitute for Active P-ADP-Rib NADP. Transferase activity was found in liver and brain microsomes and, to a smaller extent, in the cytosol fractions. In FIG. 1. Active groups in NADP. The symbols 0 and * denote Ehrlich ascites tumor cells, most of the activity resided in the specific labeling of groups in NADP species used in this study. cytosol, from which it could be partially purified. The apparent Km for phospho-ADP-ribose was about 2 x 10-4 M, and the pH ribosyltransferases or adenylyltransferases. To date, howev- optimum was around 7. Divalent cations like Mg2+ and Mn2+ er, neither phosphoADP-ribosyl- nor phosphoadenylyltrans- inhibited the reaction. In all compartmental preparations, ferases have been described, except perhaps for a nonspecific activity was eliminated by heating or short treatment with ADP-ribosyltransferase from turkey erythrocytes that can alkali or acid. In submitochondrial particles from rat liver, a release nicotinamide from NAD and NADP with similar system with different characteristics led to the phosphoadenyl- efficiency in the presence (or absence) of guanidino com- ylation of several endogenous polypeptides. pounds (4). Here we report on phosphoadenylylation as a type of Post-translational modification is used by cells to expand and enzymatic covalent modification, which differs from the to modulate properties and functions ofproteins. Apart from ATP-dependent adenylylation of prokaryotic glutamine syn- hydrolytic processes, these modifications usually require thetase, described by Stadtman and co-workers (5) and by group transferring coenzymes, in which the modifying group Holzer and co-workers (6), and also from the autoadenylyla- is present in an activated form, such as "aktivierte Es- tion of bacterial DNA ligase as an intermediate step in DNA sigsaure" in acetyl-CoA or active sulfate in adenosine 3'- ligation (7). phosphate 5'-phosphosulfate (cf. refs. 1 and 2). This applies also to ADP-ribosylation and ADP-ribosylation polymerizing reactions, in which NAD as the cosubstrate represents an MATERIALS AND METHODS activated form of ADP-ribose (ADP-Rib) (3). Labeled Compounds. Synthesis of labeled NADP from Unlike NAD, the second coenzyme NADP has been found labeled NAD (8) will be described in detail elsewhere. so far to serve exclusively oxidoreduction reactions. Here, Briefly, 4 ,umol of [3H]NAD (282 x 106 cpm/,umol) was the additional phosphate group in the 2'-hydroxyl position of incubated in 2 ml containing 20 ymol of ATP, 40 jamol of the adenine-proximal ribose is used as a signal for MgSO4, 200 Amol ofTris HCl (pH 7.5), and NAD kinase (250 dehydrogenases serving primarily anabolic reactions. How- units) at 370C for 60 min. The mixture was chromatographed ever, NADP also has two energy-rich bonds that provide an on a Dowex 1/formate column (i x 4 cm), using a linear activated adenosine 2'-phosphate, 5'-diphosphate ribose (P- gradient with 250 ml each of H20 and 4 M HCOOH. The ADP-Rib) and an activated adenosine 2',5'-bisphQsphate [3H]NADP-containing fractions were pooled and evaporated (P-AMP) residue, respectively (cf. Fig. 1). Thus, the 2'- to dryness under reduced pressure. The residue was taken up phosphate group in NADP also could serve as a discriminat- in 1 ml of H20. The yield of chromatographically pure ing signal for transferases functionally different from ADP- [3H]NADP was 90%. The publication costs of this article were defrayed in part by page charge Abbreviations: EAT, Ehrlich ascites tumor; P-ADP-Rib, adenosine payment. This article must therefore be hereby marked "advertisement" 2'-phosphate, 5'-diphosphate ribose; P-AMP, adenosine 2',5'-bis- in accordance with 18 U.S.C. §1734 solely to indicate this fact. phosphate; ADP-Rib, ADP-ribose. 6267 Downloaded by guest on September 28, 2021 6268 Biochemistry: Hilz et al. Proc. Natl. Acad. Sci. USA 83 (1986) Doubly labeled [2'-phosphate-33P, adenine-3H]NADP was buffer, pH 7.6 (18), incubated in the presence of 1 mM synthesized from [y_ 3P]ATP and [adenine-3H]NAD by in- dithiothreitol for 30 min at 37°C, and applied to 10% slab gels cubation with NAD kinase, using a modification ofthe above (sample buffer, pH 7.6). The gels were stained with Coomas- procedure (unpublished data). NAD and NADP labeled in sie blue, destained, and either dried for autoradiography other positions (cf. Fig. 1) were synthesized along the same (with the application of enhancer) or sliced (2-mm slices). To lines. [ribose(NMN)-14C]NAD was kindly provided by K. release the labeled components, slices were heated in 1 ml of Ueda (Kyoto) and by M. Jacobson (Fort Worth). 5% (vol/vol) trichloroacetic acid or 0.1 M NaOH (100'C, 60 P-[3H]ADP-Rib and P-ADP-Rib were prepared from la- min), and the radioactivity was measured. beled NADP by brief exposure to alkali (100 mM NaOH, 37TC, 10 min; M. Jacobson, personal communication), fol- lowed by chromatographic purification on Dowex 1/formate, RESULTS or by HPLC. P-AMP was obtained from Sigma or purified by Apparent PhosphoADP-Ribosyltransferase Activity in ion-exchange chromatography after treatment ofNADP with Microsomes from Rat Liver. When microsomal preparations phosphodiesterase. from rat liver were incubated with labeled NADP, a time- and Cellular Fractions and Extracts. Rat liver mitochondria, concentration-dependent incorporation of adenine equiva- mitoblasts, and submitochondrial particles were obtained as lents into the trichloroacetic acid-insoluble fraction was described (9). Extracts of submitochondrial particles were observed (Fig. 2). This activity was not caused by a phos- prepared by incubating submitochondrial particles (30 min, phatase-catalyzed breakdown of NADP to NAD and a 0C, 125 mg of protein) with 7 ml of buffer containing 6% subsequent ADP-ribosylation, because NAD was much less (vol/vol) Triton X-100, 10 mM Tris HCl, 50 mM KCl, 10 mM active than NADP in this system. Due presumably to the potassium phosphate, 1 mM EDTA, 1 mM dithiothreitol (pH trapping of labeled substrates and/or breakdown products, 7.4). The suspension was centrifuged in the cold in a Ti-75 the incorporation of adenine equivalents into the acid- Rotor at 123,000 X g for 35 min, and aliquots of the extract insoluble fraction did not exactly reflect formation of cova- were frozen until use. lently modified polypeptides as analyzed by NaDodSO4 gel Microsomes of rat liver were obtained from the post- electrophoresis. When NADP was the substrate, and the mitochondrial (12,000 x g) supernatant by centrifugation at trichloroacetic acid-insoluble reaction products were sepa- 100,000 x g for 60 min. The pellet was resuspended in 20 ml rated by NaDodSO4 gel electrophoresis and subjected to of 10 mM Hepes (pH 7.2), and aliquots were kept frozen autoradiography, one major radioactive polypeptide with an (-300C) until use. Triton X-114-extracted microsomes were apparent molecular weight of 40,000 was seen. NAD was obtained by sonication (five times for 5 sec at maximal nearly ineffective in this test. The NADP-dependent labeling output, 0C) in 0.5% purified (10) Triton X-114/10 mM ofp40 was much more pronounced in Triton X-114-extracted Hepes, pH 7, and centrifugation (100,000 x g, 60 min). The microsomes, suggesting that there were Triton-extractable pellet was resuspended in 10 mM Hepes (pH 7.2). Rat liver inhibitors (Fig. 2, Inset). nuclei were isolated as described (11). Plasma membranes Characterization of the Transferase Reaction as 2'-Phos- from mouse liver were kindly provided by E. Steinhagen- phoadenylylation of p40. The precursor used in the initial Thiessen (Hamburg, F.R.G.). experiments was a [3H]NADP synthesized from [adenine- Electrophoresis samples were precipitated with cold 10% 3H]NAD. To show incorporation of the 2'-phosphate group (vol/vol) trichloroacetic acid, washed twice with the acid and into p40, doubly labeled NADP was prepared that contained twice with ether. The dry residue was dissolved in sample a [3H]adenine moiety and a 2'-[33P]phosphate group. When A B 800- 7.5- NADP E 0 0. / 6)600- 0 92 400- .)_ 45 ._ I ,0-~ NAD U 2.5 / M _OM o ~~~~~31- 040 gOo '03 C 1) I I 1 20 40 60 20 40 Incubation time, min Microsomes, ,ul FIG. 2. Incorporation of adenine equivalents from labeled pyridine nucleotides into the microsomal acid-insoluble fraction.