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• W1966971954 abstract "The activity of protein phosphatase-1 in rat liver nuclei (PP-1N) was decreased by up to 97% by associated inhibitory polypeptides, depending on the assay and extraction conditions. These inhibitors were rapidly degraded by endogenous proteases, resulting in the accumulation of active heat-stable intermediates. Two major species of PP-1N could be differentiated by fractionation of a nuclear extract. PP-1NR111 contained, besides the δ-isoform of the catalytic subunit, an inhibitory polypeptide of 111 kDa. PP-1NR41 was found to be an inactive heterodimer between the δ-isoform of the catalytic subunit and NIPP-1, a nuclear inhibitor of PP-1, which in its undegraded form is heat labile and migrates during SDS-polyacrylamide gel electrophoresis as a polypeptide of 41 kDa. Native hepatic NIPP-1 displayed a reduced affinity for the catalytic subunit after phosphorylation by protein kinase A in vitro and after glucagon-induced phosphorylation in vivo. The activity of protein phosphatase-1 in rat liver nuclei (PP-1N) was decreased by up to 97% by associated inhibitory polypeptides, depending on the assay and extraction conditions. These inhibitors were rapidly degraded by endogenous proteases, resulting in the accumulation of active heat-stable intermediates. Two major species of PP-1N could be differentiated by fractionation of a nuclear extract. PP-1NR111 contained, besides the δ-isoform of the catalytic subunit, an inhibitory polypeptide of 111 kDa. PP-1NR41 was found to be an inactive heterodimer between the δ-isoform of the catalytic subunit and NIPP-1, a nuclear inhibitor of PP-1, which in its undegraded form is heat labile and migrates during SDS-polyacrylamide gel electrophoresis as a polypeptide of 41 kDa. Native hepatic NIPP-1 displayed a reduced affinity for the catalytic subunit after phosphorylation by protein kinase A in vitro and after glucagon-induced phosphorylation in vivo. Most processes in eukaryotic cells are controlled through phosphorylation and dephosphorylation of key proteins by protein kinases and protein phosphatases, respectively. The serine/threonine protein phosphatases of type 1 (PP-1)1 1The abbreviations used are: PP-1protein phosphatase 1PP-1Ccatalytic subunit of PP-1NIPP-1nuclear inhibitor of PP-1PP-1Nnuclear PP-1PP-1GPP-1 associated with glycogenPP-1MPP-1 associated with myofibrilsPP-1EPP-1 associated with endoplasmic reticulumPP-1Ssoluble fraction of PP-1PP-2ADdimeric form of protein phosphatase-2A, previously called PCSL or PP2A2TLCK1-chloro-3-tosylamido-7-amino-2-heptanoneTPCKL-1-tosylamido-2-phenylethyl chloromethyl ketoneDIGdigoxygeninPAGEpolyacrylamide gel electrophoresisPKAprotein kinase A. comprise an abundant and widely distributed group of enzymes that dephosphorylate proteins involved in such diverse cellular processes as metabolism, intracellular transport, protein synthesis, and cell cycle control(1Bollen M. Stalmans W. Crit. Rev. Biochem. Mol Biol. 1992; 27: 227-281Crossref PubMed Scopus (260) Google Scholar, 2Walter G. Mumby M. Biochim. Biophys. Acta. 1993; 1155: 207-226PubMed Google Scholar, 3DePaoli-Roach A.A. Park I.-K. Cerovsky V. Csortos C. Durbin S.D. Kuntz M.J. Sitikov A. Tang P.M. Verin A. Zolnierowicz S. Adv. Enzyme Regul. 1994; 34: 199-224Crossref PubMed Scopus (125) Google Scholar). They all possess an isoform of a phylogenetically very conserved catalytic subunit (PP-1C), which displays some unique properties that readily allow differentiation from other protein phosphatases. For example, only type 1 protein phosphatases are inhibited by specific cytoplasmic (inhibitor-1, inhibitor-2, DARPP-32) and nuclear (NIPP-1) polypeptides. Also, with phosphorylase as substrate, the known PP-1 holoenzymes are activated by limited trypsinolysis. This can be explained by the destruction of inhibitory noncatalytic polypeptide(s), resulting in the release of the catalytic subunit. Trypsin also hydrolyzes the carboxyl terminus of PP-1C, but this has little effect on the phosphorylase phosphatase activity. protein phosphatase 1 catalytic subunit of PP-1 nuclear inhibitor of PP-1 nuclear PP-1 PP-1 associated with glycogen PP-1 associated with myofibrils PP-1 associated with endoplasmic reticulum soluble fraction of PP-1 dimeric form of protein phosphatase-2A, previously called PCSL or PP2A2 1-chloro-3-tosylamido-7-amino-2-heptanone L-1-tosylamido-2-phenylethyl chloromethyl ketone digoxygenin polyacrylamide gel electrophoresis protein kinase A. The noncatalytic subunits of PP-1 also have a targeting function, enabling the phosphatase to associate with a particular cellular structure(1Bollen M. Stalmans W. Crit. Rev. Biochem. Mol Biol. 1992; 27: 227-281Crossref PubMed Scopus (260) Google Scholar, 2Walter G. Mumby M. Biochim. Biophys. Acta. 1993; 1155: 207-226PubMed Google Scholar, 3DePaoli-Roach A.A. Park I.-K. Cerovsky V. Csortos C. Durbin S.D. Kuntz M.J. Sitikov A. Tang P.M. Verin A. Zolnierowicz S. Adv. Enzyme Regul. 1994; 34: 199-224Crossref PubMed Scopus (125) Google Scholar, 4Bollen M. Beullens M. Van Eynde A. Stalmans W. Adv. Protein Phosphatases. 1993; 7: 31-47Google Scholar). This targeting role explains the broad subcellular distribution of PP-1 and forms the basis for the nomenclature of PP-1 holoenzymes. It is now firmly established that unique species of PP-1 are associated with glycogen (PP-1G), myofibrils (PP-1M), and in the liver also with the endoplasmic reticulum (PP-1E). A minor fraction of PP-1 is soluble (PP-1S). By far the highest concentration of PP-1, however, is found in the nucleus (PP-1N), where it can be found both in the nucleoplasm and associated with particulate structures like chromatin(4Bollen M. Beullens M. Van Eynde A. Stalmans W. Adv. Protein Phosphatases. 1993; 7: 31-47Google Scholar). Although there is convincing genetic and biochemical evidence for a role of PP-1N in nuclear processes like (alternative) pre-mRNA splicing, chromatin condensation, and gene expression, the exact substrates remain largely to be identified(4Bollen M. Beullens M. Van Eynde A. Stalmans W. Adv. Protein Phosphatases. 1993; 7: 31-47Google Scholar, 5Mermoud J.E. Cohen P.T.W. Lamond A.I. EMBO J. 1994; 13: 5679-5688Crossref PubMed Scopus (276) Google Scholar, 6Cardinali B. Cohen P.T.W. Lamond A.I. FEBS Lett. 1994; 352: 276-280Crossref PubMed Scopus (45) Google Scholar). One of the underlying problems is a complete lack of understanding of the structure and properties of native PP-1N. Although some groups reported that PP-1 was extracted from nuclei as free catalytic subunit(7Jakes S. Mellgren R.L. Schlender K.K. Biochim. Biophys. Acta. 1986; 888: 135-142Crossref PubMed Scopus (35) Google Scholar, 8Kuret J. Bell H. Cohen P. FEBS Lett. 1986; 230: 197-202Crossref Scopus (55) Google Scholar), others provided evidence for an oligomeric structure of PP-1N(4Bollen M. Beullens M. Van Eynde A. Stalmans W. Adv. Protein Phosphatases. 1993; 7: 31-47Google Scholar, 9Jessus C. Goris J. Staquet S. Cayla X. Ozon R. Merlevede W. Biochem. J. 1989; 260: 45-51Crossref PubMed Scopus (24) Google Scholar). We have purified from bovine thymus nuclei small (16-18 kDa) nuclear inhibitory polypeptides of PP-1C, termed NIPP-1(10Beullens M. Van Eynde A. Stalmans W. Bollen M. J. Biol. Chem. 1992; 267: 16538-16544Abstract Full Text PDF PubMed Google Scholar). Phosphorylation of NIPP-1 by protein kinase A and/or casein kinase 2 results in a drastic decrease in affinity for PP-1C(11Beullens M. Van Eynde A. Bollen M. Stalmans W. J. Biol. Chem. 1993; 268: 13172-13177Abstract Full Text PDF PubMed Google Scholar, 12Van Eynde A. Beullens M. Stalmans W. Bollen M. Biochem. J. 1994; 297: 447-449Crossref PubMed Scopus (34) Google Scholar). In Schizosaccharomyces pombe, a polypeptide (sds22) has been identified that is partially nuclear and acts as a positive regulator of PP-1(13Stone E.M. Yamano H. Kinoshita N. Yanagida M. Curr. Biol. 1993; 3: 13-26Abstract Full Text PDF PubMed Scopus (101) Google Scholar, 14Ohkura H. Yanagida M. Cell. 1991; 64: 149-157Abstract Full Text PDF PubMed Scopus (148) Google Scholar). It has also been demonstrated that inhibitor-2 is partially nuclear during the S-phase of the cell cycle (15Brautigan D.L. Sunwoo J. Labbé J.-C. Fernandez A. Lamb N.J.C. Nature. 1990; 344: 74-78Crossref PubMed Scopus (71) Google Scholar) and that some PP-1C is bound to the retinoblastoma protein during mitosis and early G1(16Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes & Dev. 1993; 7: 555-569Crossref PubMed Scopus (1300) Google Scholar). We have started to analyze the properties and regulation of PP-1N in rat liver nuclei and report here on the existence of at least two species of PP-1N with regulatory subunits of 41 and 111 kDa, respectively. The 41-kDa polypeptide is shown to represent the native form of hepatic NIPP-1. We also provide evidence for a physiological regulation of NIPP-1 by reversible phosphorylation. PP-1C(17DeGuzman A. Lee E.Y.C. Methods Enzymol. 1988; 159: 356-368Crossref PubMed Scopus (62) Google Scholar), PP-2AD(18Waelkens E. Goris J. Merlevede W. J. Biol. Chem. 1987; 262: 1049-1059Abstract Full Text PDF PubMed Google Scholar), inhibitor-2(19Yang S.-D. Vandenheede J.R. Merlevede W. FEBS Lett. 1981; 132: 289-295Crossref PubMed Scopus (82) Google Scholar), and phosphorylase b(20Fischer E.H. Krebs E.G. J. Biol. Chem. 1958; 231: 65-71Abstract Full Text PDF PubMed Google Scholar) were purified from rabbit skeletal muscle. Protein kinase p34cdc2 was purified from Xenopus oocytes by affinity chromatography on p13-Sepharose(21Agostinis P. Derua R. Sarno S. Goris J. Merlevede W. Eur. J. Biochem. 1992; 205: 241-248Crossref PubMed Scopus (85) Google Scholar). The recombinant α-, γ1-, γ2-, and δ-isoforms of rat PP-1C(22Zhang Z. Bai G. Shima M. Zhao S. Nagao M. Lee E.Y.C. Arch. Biochem. Biophys. 1993; 303: 402-406Crossref PubMed Scopus (61) Google Scholar) were a kind gift of Prof. E. Y. C. Lee (University of Miami, Miami, FL). Casein was prepared according to the procedure of Mercier et al.(23Mercier J.C. Maubois J.L. Poznanski S. Ribadeau-Dumas B. Bull. Soc. Chim. Biol. 1968; 50: 521-530PubMed Google Scholar). Histone IIA, myelin basic protein, catalytic subunit of PKA from beef heart and PP-2B from bovine brain were purchased from Sigma. Histone H1 and the digoxygenin (DIG) protein labeling and detection kit were obtained from Boehringer. Polyvinylidene difluoride membranes (Immobilon) were purchased from Millipore. Protein A coupled to TSK® beads, and Affi-T-agarose, used for the purification of immunoglobulins, were obtained from Affiland (Liège, Belgium). A kit for the development of Western blots by enhanced chemiluminescence was purchased from Amersham Corp. Phosphorylase b was phosphorylated in the presence of [32P]ATP by purified phosphorylase kinase(24Antoniw J.F. Nimmo H.G. Yeaman S.J. Cohen P. Biochem. J. 1977; 162: 423-433Crossref PubMed Scopus (123) Google Scholar). Casein, myelin basic protein, and histone IIA were phosphorylated by PKA(10Beullens M. Van Eynde A. Stalmans W. Bollen M. J. Biol. Chem. 1992; 267: 16538-16544Abstract Full Text PDF PubMed Google Scholar), and histone H1 was phosphorylated by p34cdc2(21Agostinis P. Derua R. Sarno S. Goris J. Merlevede W. Eur. J. Biochem. 1992; 205: 241-248Crossref PubMed Scopus (85) Google Scholar). For the preparation of digoxygenin-labeled PP-1C, 200 μl of purified catalytic subunit (0.5 mg/ml) was dialyzed against 50 mM sodium tetraborate at pH 8.5 plus 1 mM dithiothreitol, and incubated with 15 μl of digoxygenin-carboxymethyl-N-hydroxysuccinimide ester for 1 h at room temperature. Subsequently, the mixture was extensively dialyzed against a solution containing 20 mM Tris-HCl at pH 7.5, 1 mM dithiothreitol, and 60% glycerol. All experiments were performed with normally fed female Wistar rats of about 250 g. Unless indicated otherwise, the animals were sacrificed by decapitation. To check whether phosphorylation of NIPP-1 by PKA also occurs in vivo (see Fig. 7), the animals were first injected intraperitoneally with propranolol (1 mg in 0.2 ml of saline) plus glucose (1 g in 3 ml of saline). 30 min later, the activity of liver PKA was enhanced by the intraperitoneal injection of glucagon (0.3 mg in 0.2 ml of saline). 15 min before excision of the liver, the animals were anesthetized by an intraperitoneal injection of sodium pentobarbital (12 mg in 0.2 ml of saline). In this experiment, nuclei were prepared in the presence of 40 mM NaF and 0.2 μM microcystin to prevent dephosphorylation of NIPP-1. Unless indicated otherwise, all purification and fractionation buffers were supplemented with a mixture of protease inhibitors including 0.5 mM phenylmethanesulfonyl fluoride, 50 μM 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK), 50 μML-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK), and 5 μM leupeptin. Liver nuclei were prepared according to the method of Blobel and Potter(25Blobel G. Potter V.R. Science. 1966; 154: 1662-1665Crossref PubMed Scopus (986) Google Scholar), with slight modifications. Briefly, livers were homogenized in 2 volumes of buffer A containing 15 mM PIPES/NaOH at pH 7.2, 80 mM KCl, 15 mM NaCl, 0.5 mM dithiothreitol, and 0.25 M sucrose. The homogenate was mixed with 2 volumes of buffer A containing 2.3 M instead of 0.25 M sucrose, and put on top of 1 volume of the same buffer. After high speed centrifugation (120,000 × g for 30 min), the pelleted nuclei were resuspended with a Dounce homogenizer (A pestle) in buffer B, containing 20 mM Tris-HCl at pH 7.4, 1 mM MgCl2, 0.25 M sucrose, and 0.5% Triton X-100. After standing on ice for 10 min, enabling the complete solubilization of the outer nuclear membrane, the nuclear suspension was centrifuged for 5 min at 3,500 × g. The nuclear pellet was homogenized with a Dounce homogenizer (B pestle) in a hypotonic solution (buffer C) containing 50 mM glycylglycine/NaOH at pH 7.4, 5 mM 2-mercaptoethanol, and 0.5 mM dithiothreitol, and the lysed nuclei were submitted to centrifugation during 5 min at 13,000 × g. The pellet was resuspended in buffer C plus 0.3 M NaCl, incubated for 30 min at 4°C, and centrifuged during 5 min at 13,000 × g. The latter supernatant is further referred to as “nuclear extract.” The protocol was based on the procedure described by Alessi et al.(26Alessi D. Macdougall L.K. Sola M.M. Ikebe M. Cohen P. Eur. J. Biochem. 1992; 210: 1023-1035Crossref PubMed Scopus (331) Google Scholar). Briefly, following SDS-PAGE (10%), the separated polypeptides were blotted onto Immobilon membranes and incubated for 3 h at room temperature with a buffer containing 50 mM Tris-HCl at pH 7.5, 0.5 M NaCl, and 5% skim milk powder. Subsequently, the blots were incubated for 3 h in 10 mM Tris-HCl at pH 7.4, 150 mM NaCl, 1 mg/ml bovine serum albumin, and a 1:1000 dilution of DIG-labeled PP-1C. Finally, the free DIG-PP-1C was washed away, and the bound phosphatase was detected with DIG-specific antibodies according to the manufacturer's protocol. Rabbit polyclonal antibodies against a synthetic peptide encompassing the carboxyl-terminal domain (residues 302-322) of the δ-isoform of PP-1C were kindly donated by Prof. A. A. DePaoli-Roach (Indiana University, Indianapolis, IN). Antibodies against a synthetic peptide encompassing the 11 carboxyl-terminal residues of NIPP-1 from bovine thymus were raised in a rabbit and purified by chromatography on Affi-T-agarose. For Western blotting, incubation with the antisera was done for 2 h at room temperature at a final dilution of 1:2000, and the peroxidase-labeled secondary antibodies were detected by enhanced chemiluminescence. For immunoprecipitation of PP-1NR41 and PP-1NR111, 300 μl of the Mono Q peak fractions (see “Results”) were first incubated with the PP-1CS antiserum (final dilution of 1:60) or with Affi-T-purified NIPP-1 antibodies (final concentration of 30 μg/ml) during 45 min at 4°C. Subsequently, 30 μl of protein-A-TSK® was added, and the mixture was rotated during 30 min at 4°C. Following centrifugation (1 min at 10,000 × g), the pellet was first washed in 0.5 ml of a buffer containing 10 mM Tris-HCl at pH 7.5, 1 mM dithiothreitol, and 0.25 M LiCl and then washed twice in 1 ml of the same buffer without LiCl. The supernatant and resuspended pellet were assayed for spontaneous and trypsin-revealed phosphorylase phosphatase activities. An aliquot of these fractions was boiled in SDS-sample buffer and centrifuged (1 min at 10,000 × g), and the supernatant was checked for binding of DIG-PP-1C following denaturing electrophoresis and blotting. The spontaneous and trypsin-revealed protein phosphatase activities were measured as described previously(10Beullens M. Van Eynde A. Stalmans W. Bollen M. J. Biol. Chem. 1992; 267: 16538-16544Abstract Full Text PDF PubMed Google Scholar). One unit of phosphatase releases 1 nmol of phosphate/min at 30°C. Heat-stable inhibitory polypeptides of PP-1C were assayed using 32P-labeled phosphorylase a as substrate. Following a heat treatment (3 min at 90°C), the inhibitory activity was assayed before and after preincubation of the fractions with trypsin (0.1 mg/ml during 5 min). The difference between both assays was taken as protein-derived inhibitory activity. Since all known species of PP-1 can dephosphorylate phosphorylase a, we have chosen this substrate for the initial characterization of PP-1N. Although the phosphorylase phosphatase activity was very low in freshly prepared liver nuclei, the activity was increased up to 40-fold by a preincubation of the nuclear fraction with trypsin (Fig. 1), which is known to release the catalytic subunit (see the Introduction). Both, the spontaneous and the trypsin-revealed activities could be nearly completely blocked by the addition of inhibitor-2 (not illustrated), showing that the activity stemmed virtually exclusively from PP-1. A large activation (5-25-fold) of PP-1N by trypsinolysis was also obtained after prior extraction of PP-1N with 0.3 M NaCl (see below). The spontaneous phosphorylase phosphatase activity gradually increased during incubation of the nuclei at 37°C in the presence of phenylmethanesulfonyl fluoride as sole protease inhibitor (Fig. 1). This increase was completely blocked by the addition of the protease inhibitors leupeptin, TPCK, and TLCK. In separate experiments, it was found that the addition of leupeptin alone was able to prevent completely the time-dependent increase in the spontaneous activity of PP-1N, while the addition of TPCK or TLCK was only partially effective (not illustrated). On the other hand, the trypsin-revealed phosphorylase phosphatase activity, which is a measure for the concentration of the catalytic subunit, remained constant during incubation with phenylmethanesulfonyl fluoride only. Since we had previously identified heat-stable inhibitory polypeptides of PP-1 (NIPP-1) in thymus nuclei(10Beullens M. Van Eynde A. Stalmans W. Bollen M. J. Biol. Chem. 1992; 267: 16538-16544Abstract Full Text PDF PubMed Google Scholar), we wondered whether the presence of such inhibitor(s) could account for the low spontaneous activity of hepatic PP-1N. To our surprise, however, we found that freshly prepared liver nuclei contained very little or no heat-stable inhibitory activity of PP-1C, although such an inhibitory activity could be generated by simple incubation of the nuclei at 37°C (Fig. 2). Again, the generation of this heat-stable inhibitory activity was completely blocked by the addition of the protease inhibitors leupeptin, TLCK, and TPCK. Taken together, the above data suggest that the activity of PP-1C in the nucleus is suppressed by polypeptide(s) that are destroyed by trypsin. These inhibitory polypeptides seem also to be degraded by endogenous protease(s), which explains the gradual increase of the spontaneous phosphorylase phosphatase activity (Fig. 1). However, the endogenous proteases also generate some active heatstable inhibitor fragments (Fig. 2). Upon chromatography of a nuclear salt extract on Mono Q, the phosphorylase phosphatase activity eluted in two major peaks (Fig. 3A). The first peak, termed PP-1NR41, was nearly completely latent, but the activity could be revealed by a preincubation with trypsin, which results in the release of free catalytic subunit (see the Introduction). The second peak (PP-1NR111) was always partially active but could be additionally activated up to 5-fold by trypsin. The extent of activation of PP-1NR111 by trypsin varied among preparations and moreover decreased rapidly during storage of the fractions on ice or at −20°C. Although the addition of leupeptin, TPCK, and TLCK blocked the degradation of PP-1N during purification and incubation of liver nuclei (Fig. 1), these inhibitors failed to block proteolysis during further purification of PP-1N. Thus, efforts to purify PP-1NR41 and PP-1NR111 until homogeneity failed and invariably resulted in the generation of free catalytic subunit. Problems with proteolysis were particularly pronounced for PP-1NR111 (see also below). For the identification of the noncatalytic subunits of PP-1N, we have therefore used an indirect method that is based upon the high affinity binding of DIG-labeled PP-1C to specific polypeptides, following SDS-PAGE (10%), and blotting of a nuclear extract. Using this approach, we could identify two major PP-1C-binding polypeptides of 41 ± 1 (n = 12) and 111 ± 2 kDa (n = 7), that coeluted during chromatraphy on Mono Q with PP-1NR41 and PP-1NR111, respectively (Fig. 3B). That the 41- and 111-kDa polypeptides, further denoted as R41 and R111, were physically associated with the catalytic subunit is substantiated by their co-immunoprecipitation with PP-1C, using polyclonal antibodies against the C-terminal domain of δ-isoform of the catalytic subunit (Fig. 3C). In agreement with the extreme sensitivity of R111 to proteolysis, we noted that this polypeptide was partially degraded to smaller fragments during immunoprecipitation which, however, were still able to bind DIG-PP-1C (Fig. 3C, lane2). It should also be noted that the immunoprecipitated PP-1N holoenzymes were still largely latent, as indicated by the severalfold increase of the phosphorylase phosphatase activity by a preincubation with trypsin (not illustrated). Since the recent cDNA cloning of NIPP-1 revealed that the inhibitor(s) purified from bovine thymus nuclei (16-18 kDa) were proteolytic fragments and that recombinant NIPP-1 migrated during SDS-PAGE (10%) as a protein of 41 kDa,2 2A. Van Eynde, S. Wera, M. Beullens, S. Torrekens, F. Van Leuven, W. Stalmans, and M. Bollen, unpublished data. we wondered whether R41 could be identical to NIPP-1. We indeed noted that in the Mono Q fractions containing PP-1NR41, a polypeptide of 41 kDa was detected by Western blotting with polyclonal antibodies against the C-terminal domain of NIPP-1 (not shown). This 41-kDa polypeptide was no longer detected when the primary antibodies were preincubated with an excess of the synthetic peptide to which the antibodies were generated. As is illustrated in Fig. 4, PP-1NR41 could be nearly quantitatively immunoprecipated with the NIPP-1 antibodies and in the immunoprecipitate R41 could be identified by labeling with DIG-PP-1C. Gel filtration on Superdex 75 showed that PP-1NR41 migrated as a protein of about 90 kDa (Fig. 5A). Combined with the above data, these results suggest that PP-1NR41 is a heterodimer between the catalytic subunit (37 kDa, see below) and NIPP-1. Gel filtration of PP-1NR111 always yielded at least two peaks of activity with apparent molecular masses of about 160 and 30-40 kDa (Fig. 5B). The first peak was largely latent and may represent a heterodimer of the catalytic subunit and R111. The second peak was fully active and is likely to be the free catalytic subunit, generated by proteolysis or dissociation of the noncatalytic subunit(s). The latter view is substantiated by findings that gel filtration of stored samples of PP-1NR111 exclusively yielded free catalytic subunit (not illustrated). There are five known mammalian isoforms of PP-1C(1Bollen M. Stalmans W. Crit. Rev. Biochem. Mol Biol. 1992; 27: 227-281Crossref PubMed Scopus (260) Google Scholar, 2Walter G. Mumby M. Biochim. Biophys. Acta. 1993; 1155: 207-226PubMed Google Scholar, 3DePaoli-Roach A.A. Park I.-K. Cerovsky V. Csortos C. Durbin S.D. Kuntz M.J. Sitikov A. Tang P.M. Verin A. Zolnierowicz S. Adv. Enzyme Regul. 1994; 34: 199-224Crossref PubMed Scopus (125) Google Scholar, 16Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes & Dev. 1993; 7: 555-569Crossref PubMed Scopus (1300) Google Scholar), and it has been demonstrated that the α-, γ1-, and δ-isoforms are present in rat liver(27Goodwin G.H. Sanders C. Johns E.W. Eur. J. Biochem. 1973; 38: 14-19Crossref PubMed Scopus (600) Google Scholar). Western analysis with antibodies that rather specifically recognized the δ-isoform of PP-1C yielded a signal for both PP-1NR41 and PP-1NR111 (Fig. 6). The presence of the δ-isoform is corroborated by findings that the two holoenzymes could be immunoprecipitated with these antibodies (Fig. 3C). Surprisingly, the catalytic subunit of PP-1NR111 migrated as a slightly larger protein than recombinant PP-1CS and than the catalytic subunit of PP-1NR41. These differences may reflect a distinct posttranslational modification or the presence of two δ-isoforms generated, for example, by alternative splicing. Along the same line, it has been shown that there are two forms of PP-1Cα that only differ by the presence or absence of an amino-terminal 11-amino acid insert(16Durfee T. Becherer K. Chen P.-L. Yeh S.-H. Yang Y. Kilburn A.E. Lee W.-H. Elledge S.J. Genes & Dev. 1993; 7: 555-569Crossref PubMed Scopus (1300) Google Scholar). The above data imply that the noncatalytic subunit(s) of PP-1NR41 and PP-1NR111 are inhibitory, using phosphorylase as substrate. Since the known regulatory subunits of the cytoplasmic species of PP-1 can be either inhibitory or stimulatory, depending on the substrate (1Bollen M. Stalmans W. Crit. Rev. Biochem. Mol Biol. 1992; 27: 227-281Crossref PubMed Scopus (260) Google Scholar, 2Walter G. Mumby M. Biochim. Biophys. Acta. 1993; 1155: 207-226PubMed Google Scholar, 3DePaoli-Roach A.A. Park I.-K. Cerovsky V. Csortos C. Durbin S.D. Kuntz M.J. Sitikov A. Tang P.M. Verin A. Zolnierowicz S. Adv. Enzyme Regul. 1994; 34: 199-224Crossref PubMed Scopus (125) Google Scholar), we have investigated whether this also applies to PP-1N. For that purpose, we have compared the effect of trypsinolysis of the nuclear holoenzymes on their activity toward various substrates (Table 1). Depending on the substrate, trypsinolysis of PP-1NR41 increased the phosphatase activity 2-13-fold. On the other hand, a preincubation of PP-1NR111 with trypsin only caused a 2-5-fold increase in the phosphatase activities toward phosphorylase, myelin basic protein, and histone H1 and had no effect on the casein and histone IIA phosphatase activities. Table 1 also shows that the activity of the catalytic subunit toward all investigated substrates was either not affected or only slightly increased by trypsinolysis, implying that the observed effects of trypsin on the nuclear holoenzymes are largely or completely due to the destruction of the noncatalytic subunit(s). In conclusion, our data suggest that NIPP-1 and R111 are, if anything, inhibitory to the dephosphorylation of the investigated substrates.TABLE I Open table in a new tab We have previously shown that the active fragments of NIPP-1 from bovine thymus show a decreased affinity for PP-1C after phosphorylation by protein kinase A or casein kinase-2(11Beullens M. Van Eynde A. Bollen M. Stalmans W. J. Biol. Chem. 1993; 268: 13172-13177Abstract Full Text PDF PubMed Google Scholar, 12Van Eynde A. Beullens M. Stalmans W. Bollen M. Biochem. J. 1994; 297: 447-449Crossref PubMed Scopus (34) Google Scholar). In agreement with this result we found that after incubation of hepatic PP-1NR41 with PKA under phosphorylating conditions and subsequent denaturing electrophoresis, NIPP-1 reproducibly bound less DIG-labeled catalytic subunit (Fig. 7, upperleftpanel). Interestingly, the remaining label was bound to a polypeptide, which migrated somewhat slower during SDS-PAGE, indicating that the electrophoretic mobility of NIPP-1 is decreased by phosphorylation with PKA. A lesser DIG-PP-1C labeling was also obtained after phosphorylation of blotted NIPP-1 with PKA. Subsequent incubation with PP-2AD and PP-2B restored binding of DIG-PP-1C, providing additional evidence that the observed effect was due to phosphorylation of NIPP-1 and not, for example, due to a binding of PKA or a (proteolytic) loss of NIPP-1. Incubation of PP-1NR41 or blotted hepatic NIPP-1 with casein kinase-2 under phosphorylating conditions did not affect the extent of binding of DIG-PP-1C to NIPP-1 (not shown). Although phosphorylation of native PP-1NR41 by PKA decreased the affinity of NIPP-1 for DIG-PP-1C, it did not affect the phosphorylase phosphatase activity (not shown), suggesting that the decrease in the affinity was not sufficient to cause a dissociation of the holoenzyme. However, a 2-4-fold increase in the phosphorylase phosphatase activity was detected after phosphorylation of partially purified PP-1NR41 with PKA or casein kinase-2 (not shown). This partially purified enzyme was obtained by consecutive chromatographies of a nuclear extract on Mono Q, heparin-Sepharose, and histone IIA-Sepharose (not illustrated), and was almost certainly a proteolyzed species, as indicated by observations that the associated NIPP-1 inhibitory activity was heat stable. We have also used the DIG-PP-1C-labeling method to investigate whether NIPP-1 is also a substrate for PKA in vivo. For that purpose, rats were injected for various times with glucagon after which liver nuclei were prepared in the presence of phosphatase inhibitors to prevent dephosphorylation of NIPP-1. In the lowerpanel of Fig. 7, it is shown that the administration of glucagon resulted in a time-dependent decrease of the binding of DIG-PP-1C to NIPP-1, with a maximal effect after 30-60 min. By 90 min the binding of DIG-PP-1C had almost returned to the control value. Again, all of the observed differences were abolished by preincubation of the blots with a mixture of PP-2AD and PP-2B. Also, Western analysis showed an equal concentration of NIPP-1 at all investigated time points (not shown), providing additional evidence that the observed differences cannot be explained by a different loading of NIPP-1. We did not find any evidence for a regulation of the activity of PP-1NR111 or of the binding of DIG-PP-1C to R111 through phosphorylation by PKA or casein kinase-2. In two previous studies it was concluded that PP-1 is present in hepatic nuclear extracts as free catalytic subunit(7Jakes S. Mellgren R.L. Schlender K.K. Biochim. Biophys. Acta. 1986; 888: 135-142Crossref PubMed Scopus (35) Google Scholar, 8Kuret J. Bell H. Cohen P. FEBS Lett. 1986; 230: 197-202Crossref Scopus (55) Google Scholar). This contrasts with the data of the present work, showing an association of the catalytic subunit with inhibitory polypeptides. One obvious reason for this discrepancy is our use of lower NaCl concentrations (0.3 M) for the solubilization of PP-1N versus 1-2 M in (7Jakes S. Mellgren R.L. Schlender K.K. Biochim. Biophys. Acta. 1986; 888: 135-142Crossref PubMed Scopus (35) Google Scholar) and (8Kuret J. Bell H. Cohen P. FEBS Lett. 1986; 230: 197-202Crossref Scopus (55) Google Scholar), which presumably caused dissociation of the catalytic subunit. Moreover, at NaCl concentrations above 0.3 M, histones are extracted(27Goodwin G.H. Sanders C. Johns E.W. Eur. J. Biochem. 1973; 38: 14-19Crossref PubMed Scopus (600) Google Scholar), which have been shown to interfere with the phosphorylase phosphatase assay(1Bollen M. Stalmans W. Crit. Rev. Biochem. Mol Biol. 1992; 27: 227-281Crossref PubMed Scopus (260) Google Scholar). An additional explanation for the generation of free PP-1C in nuclear fractions is the extreme sensitivity of regulatory polypeptides to endogenous proteases (Figure 1:, Figure 2:). Perhaps such proteolysis is mediated by the multicatalytic proteasome complex, which was shown to be present in liver nuclei(28Rivett A.J. Biochem. J. 1993; 291: 1-10Crossref PubMed Scopus (383) Google Scholar). In agreement with this proposal, we found that the degradation of the regulatory polypeptides of PP-1 is blocked by leupeptin (Figure 1:, Figure 2:), which has also been found to block the trypsin-like activity associated with proteasomes. It cannot be excluded that nuclear proteolysis provides a mechanism for regulating the activity of PP-1N in vivo, as has been shown for other nuclear proteins(29Cressman D.E. Taub R. J. Biol. Chem. 1994; 269: 26594-26597Abstract Full Text PDF PubMed Google Scholar). A widely used approach for the initial identification of the protein phosphatase(s) that dephosphorylate a particular substrate is based on the screening of subcellular fractions in the presence or absence of specific phosphatase inhibitors/activators(1Bollen M. Stalmans W. Crit. Rev. Biochem. Mol Biol. 1992; 27: 227-281Crossref PubMed Scopus (260) Google Scholar). Such a strategy has for example recently been employed for the identification of the nuclear phosphatase(s) acting on p34cdc2-phosphorylated histone H1 (30Sola M.M. Langan T. Cohen P. Biochim. Biophys. Acta. 1991; 1094: 211-216Crossref PubMed Scopus (57) Google Scholar) or PKA-phosphorylated cAMP response element binding protein, CREB(31Wadzinski B.E. Wheat W.H. Jaspers S. Peruski Jr., L.F. Lickteig R.L. Johnson G.L. Klemm D.J. Mol. Cell. Biol. 1993; 13: 2822-2834Crossref PubMed Scopus (287) Google Scholar). Our investigations show that this approach may yield a wrong estimate for the contribution of PP-1N. Indeed, unless a host of protease inhibitors are added and fresh material is used, one obtains free catalytic subunit whose substrate specificity differs from that of holoenzymes (Table 1). It is also important to note that PP-1N itself is controlled by reversible phosphorylation (this study and Refs. 11 and 12). In the absence of functional protein kinases (for lack of MgATP), PP-1N will be (auto)dephosphorylated during tissue fractionation, and studies on such fractions may therefore not be informative for the activity of native PP-1N. Due to the extreme sensitivity of the noncatalytic polypeptides of PP-1 to proteolysis (this study and (1Bollen M. Stalmans W. Crit. Rev. Biochem. Mol Biol. 1992; 27: 227-281Crossref PubMed Scopus (260) Google Scholar)), the PP-1 holoenzymes are very resistant to purification. We have therefore adopted an alternative strategy for the identification of the noncatalytic subunits in crude fractions that is based upon their high affinity binding of DIG-labeled PP-1C (Fig. 3). The same approach has previously been used for the identification of the PP-1C-binding subunit of purified PP-1M(26Alessi D. Macdougall L.K. Sola M.M. Ikebe M. Cohen P. Eur. J. Biochem. 1992; 210: 1023-1035Crossref PubMed Scopus (331) Google Scholar). It should be noted, however, that this approach does not necessarily allow one to identify all PP-1C-binding polypeptides. Thus, we have noted that inhibitor 2 and the active 16-18-kDa fragments of NIPP-1 from bovine thymus do not bind DIG-PP-1C after denaturing electrophoresis. Also, although it seems likely that the heat-stable inhibitor(s) of PP-1C that are generated during incubation of nuclei (Fig. 2) are proteolytic degradation products of NIPP-1 and/or R111, we have been unable to detect the accumulation of small, heat-stable inhibitor(s) with the DIG-PP-1C-labeling technique (not shown). The copurification and coimmunoprecipitation of NIPP-1 and PP-1NR41 (Fig. 3-5), combined with the migration of the holoenzyme during gel filtration as a protein of about 90 kDa (Fig. 5A), strongly indicate that native PP-1NR41 is a heterodimer of PP-1C and NIPP-1. We suggest that PP-1NR41 represents the native hepatic homolog of PP-1Nα, which was previously identified in thymus nuclear extracts(11Beullens M. Van Eynde A. Bollen M. Stalmans W. J. Biol. Chem. 1993; 268: 13172-13177Abstract Full Text PDF PubMed Google Scholar, 12Van Eynde A. Beullens M. Stalmans W. Bollen M. Biochem. J. 1994; 297: 447-449Crossref PubMed Scopus (34) Google Scholar). The latter migrated as a 50-kDa polypeptide during gel filtration and was also proposed to be a heterodimer of the catalytic subunit and NIPP-1. The smaller mass of the thymus enzyme is likely to be explained by proteolysis of NIPP-1 during nuclear fractionation resulting in the generation of active 16-18-kDa fragments. The copurification and coimmunoprecipitation of R111 and PP-1NR111 (Fig. 3) suggest that R111 is a subunit of PP-1NR111. From gel filtration, we deduced an apparent molecular mass of about 160 kDa, which agrees with a heterodimeric structure between PP-1C and R111. However, due to the extreme lability of the enzyme (Fig. 4B), it cannot be excluded that the native phosphatase is larger and/or contains additional subunit(s). Western analysis suggested that both PP-1NR41 and PP-1NR111 contain the δ-isoform of the catalytic subunit (Fig. 6). Moreover, both species of PP-1N could be immunoprecipitated with PP-1CS-specific antibodies. However, since only about 70% of the activities of PP-1NR41 and PP-1NR111 were precipitated with these antibodies, it cannot be ruled out that a minor fraction of the holoenzymes contains other isoform(s) of PP-1C. We found that the phosphorylation of NIPP-1 by PKA was associated with a lesser binding of PP-1C (Fig. 7). This agrees with similar findings on PP-1Nα from bovine thymus(11Beullens M. Van Eynde A. Bollen M. Stalmans W. J. Biol. Chem. 1993; 268: 13172-13177Abstract Full Text PDF PubMed Google Scholar). However, in contrast with the thymus enzyme and with the partially purified liver enzyme, which both contain a heat-stable proteolytic fragment of NIPP-1, intact hepatic PP-1NR41 was not activated by phosphorylation with PKA and/or casein kinase 2. This suggests that native NIPP-1 is more tightly bound to the catalytic subunit than are the heat-stable fragments of NIPP-1. Perhaps, phosphorylation by additional protein kinases is required for dissociation of the native holoenzyme. It can also be envisaged that dissociation and reassociation of PP-1NR41 are independently controlled processes and that PKA only controls the reassociation of the holoenzyme. We thank Dr. A. A. DePaoli-Roach for the generous gift of polyclonal antibodies against PP-1CS and Dr. E. Y. C Lee for the recombinant isoforms of PP-1C. The p34cdc2 used in this study was a gift of Drs. R. Derua and J. Goris. We thank Dr. J. R. Vandenheede for advice on the immunoprecipitation of PP-1N and Dr. S. Wera for comments on the manuscript. Nicole Sente and Peter Vermaelen provided expert technical assistance." @default.
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