©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Subunit Structure and Regulation of Protein Phosphatase-1 in Rat Liver Nuclei (*)

(Received for publication, March 10, 1995; and in revised form, May 8, 1995)

Izabela Jagiello (§) , Monique Beullens , Willy Stalmans , Mathieu Bollen (¶)

From the Afdeling Biochemie, Faculteit Geneeskunde, Katholieke Universiteit Leuven, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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-1N contained, besides the -isoform of the catalytic subunit, an inhibitory polypeptide of 111 kDa. PP-1N was found to be an inactive heterodimer between the -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.


INTRODUCTION

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)()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(1, 2, 3) . They all possess an isoform of a phylogenetically very conserved catalytic subunit (PP-1), 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-1, but this has little effect on the phosphorylase phosphatase activity.

The noncatalytic subunits of PP-1 also have a targeting function, enabling the phosphatase to associate with a particular cellular structure(1, 2, 3, 4) . 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(4) .

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(4, 5, 6) . 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(7, 8) , others provided evidence for an oligomeric structure of PP-1N(4, 9) . We have purified from bovine thymus nuclei small (16-18 kDa) nuclear inhibitory polypeptides of PP-1, termed NIPP-1(10) . Phosphorylation of NIPP-1 by protein kinase A and/or casein kinase 2 results in a drastic decrease in affinity for PP-1(11, 12) . In Schizosaccharomyces pombe, a polypeptide (sds22) has been identified that is partially nuclear and acts as a positive regulator of PP-1(13, 14) . It has also been demonstrated that inhibitor-2 is partially nuclear during the S-phase of the cell cycle (15) and that some PP-1 is bound to the retinoblastoma protein during mitosis and early G(16) .

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.


EXPERIMENTAL PROCEDURES

Materials

PP-1(17) , PP-2A(18) , inhibitor-2(19) , and phosphorylase b(20) were purified from rabbit skeletal muscle. Protein kinase p34 was purified from Xenopus oocytes by affinity chromatography on p13-Sepharose(21) . The recombinant -, -, -, and -isoforms of rat PP-1(22) 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.(23) . 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 [P]ATP by purified phosphorylase kinase(24) . Casein, myelin basic protein, and histone IIA were phosphorylated by PKA(10) , and histone H1 was phosphorylated by p34(21) .

For the preparation of digoxygenin-labeled PP-1, 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.

Treatment of the Animals

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.


Figure 7: Reduced binding of DIG-PP-1 after phosphorylation of hepatic NIPP-1 by PKA. An aliquot of the Mono Q peak fraction of PP-1N was incubated during 60 min at 30 °C in buffer C plus 0.2 mM ATP, 2 mM magnesium acetate, 1 mg/ml bovine serum albumin in the absence or presence of 80 nM PKA. Following electrophoresis and blotting, the membranes were screened for binding of DIG-PP-1 (upperleftpanel). Other samples of PP-1N were first subjected to denaturing electrophoresis and blotting. Subsequently, the latter blots were incubated under phosphorylation conditions in the absence or presence of PKA, which was followed by an incubation during 120 min at room temperature in buffer C, 1 mg/ml bovine serum albumin in the absence or presence of PP-2A (2 units/ml) and PP-2B (25 units/ml). Finally, the blots were labeled with DIG-PP-1 (upperrightpanel). The lowerleftpanel shows the binding of DIG-PP-1 to NIPP-1 in hepatic nuclear extracts prepared from rats that were treated for the indicated times with glucagon, as detailed under ``Experimental Procedures.'' The lowerrightpanel shows DIG-PP-1 binding to the same samples after preincubation of the blots with PP-2A plus PP-2B.



Purification of Nuclei and Subnuclear Fractionation

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(25) , 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 MgCl, 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.''

Affinity Labeling with Digoxygenin-bound PP-1

The protocol was based on the procedure described by Alessi et al.(26) . 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-1. Finally, the free DIG-PP-1 was washed away, and the bound phosphatase was detected with DIG-specific antibodies according to the manufacturer's protocol.

Western Blotting and Immunoprecipitation

Rabbit polyclonal antibodies against a synthetic peptide encompassing the carboxyl-terminal domain (residues 302-322) of the -isoform of PP-1 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-1N and PP-1N, 300 µl of the Mono Q peak fractions (see ``Results'') were first incubated with the PP-1 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-1 following denaturing electrophoresis and blotting.

Assays

The spontaneous and trypsin-revealed protein phosphatase activities were measured as described previously(10) . One unit of phosphatase releases 1 nmol of phosphate/min at 30 °C. Heat-stable inhibitory polypeptides of PP-1 were assayed using P-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.


RESULTS

Hepatic PP-1N Is a Largely Latent Enzyme

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).


Figure 1: Proteolytic activation of PP-1N during incubation of nuclei. Nuclei were prepared in the presence of either 0.5 mM phenylmethanesulfonyl fluoride as sole protease inhibitor (, ) or in the presence of 0.5 mM phenylmethanesulfonyl fluoride, 50 µM TLCK, 50 µM TPCK, and 5 µM leupeptin (). The nuclei (10 10/ml) were incubated at 37 °C in 20 mM Tris-HCl at pH 7.5, 0.3 M sucrose, 5 mM MgCl, 2 mM EDTA, 2.5 mM CaCl and the same mixture of protease inhibitors. At the indicated times, samples were taken for the assay of spontaneous (opensymbols) and trypsin-revealed () phosphorylase phosphatase activities. For the assay, the samples were 30-fold diluted in a hypotonic medium. The results represent the means ± S.E. for three different preparations.



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(10) , 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-1, 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-1 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).


Figure 2: Proteolytic generation of heat-stable inhibitor(s) of PP-1. From the same nuclear suspensions that were used for the assay of phosphorylase phosphatase (Fig. 1), samples were taken for the assay of heat-stable inhibitory protein, as indicated under ``Experimental Procedures.'' The nuclear suspension had been prepared and incubated in the absence () or presence () of TPCK, TLCK, and leupeptin. The results represent the means ± S.E. for three different preparations.



Identification of Two Species of PP-1N

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-1N, 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-1N) was always partially active but could be additionally activated up to 5-fold by trypsin. The extent of activation of PP-1N by trypsin varied among preparations and moreover decreased rapidly during storage of the fractions on ice or at -20 °C.


Figure 3: Fractionation of PP-1N on Mono Q. A freshly prepared nuclear extract from the livers of two rats was dialyzed against buffer C and was applied to a Mono Q column, equilibrated in the same buffer. Bound proteins were eluted with a linear salt gradient (0-0.4 M NaCl). The fractions were assayed for spontaneous () and trypsin-revealed () phosphorylase phosphatase activities (A) and for binding of DIG-PP-1 after SDS-PAGE (10%) and blotting onto Immobilon membranes (B). The peak fractions of PP-1N and PP-1N were also used for immunoprecipitation with antibodies against PP-1. PanelC shows the labeling of the immunoprecipitates with DIG-PP-1, performed as in panelB.



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-1N and PP-1N until homogeneity failed and invariably resulted in the generation of free catalytic subunit. Problems with proteolysis were particularly pronounced for PP-1N (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-1 to specific polypeptides, following SDS-PAGE (10%), and blotting of a nuclear extract. Using this approach, we could identify two major PP-1-binding polypeptides of 41 ± 1 (n = 12) and 111 ± 2 kDa (n = 7), that coeluted during chromatraphy on Mono Q with PP-1N and PP-1N, 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-1, using polyclonal antibodies against the C-terminal domain of -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-1 (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).

Identification of R41 as NIPP-1

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,()we wondered whether R41 could be identical to NIPP-1. We indeed noted that in the Mono Q fractions containing PP-1N, 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-1N could be nearly quantitatively immunoprecipated with the NIPP-1 antibodies and in the immunoprecipitate R41 could be identified by labeling with DIG-PP-1.


Figure 4: Immunoprecipitation of PP-1N with NIPP-1 antibodies. The peak fractions of Mono Q containing PP-1N were pooled and tested for immunoprecipation with NIPP-1 antibodies. The immunoprecipitate was resuspended until the same volume as the supernatant, and both fractions were assayed for spontaneous and trypsin-revealed phosphorylase phosphatase activities (A). PanelB shows the labeling of the supernatant and the immunoprecipitate with DIG-PP-1 as in Fig. 3C.



Subunit Structure of PP-1N

Gel filtration on Superdex 75 showed that PP-1N migrated as a protein of about 90 kDa (Fig. 5A). Combined with the above data, these results suggest that PP-1N is a heterodimer between the catalytic subunit (37 kDa, see below) and NIPP-1. Gel filtration of PP-1N 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-1N exclusively yielded free catalytic subunit (not illustrated).


Figure 5: Gel filtration of PP-1N. 200 µl of the Mono Q pool of PP-1N (upperpanel) or PP-1N (lowerpanel) were applied to a Superdex-75 column, equilibrated in buffer C plus 0.1 M NaCl. The fractions were assayed for spontaneous () and trypsin-revealed () phosphorylase phosphatase activities. The arrows indicate the elution position of marker proteins, i.e. phosphorylase (185 kDa), aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and PP-1 (37 kDa).



There are five known mammalian isoforms of PP-1(1, 2, 3, 16) , and it has been demonstrated that the -, -, and -isoforms are present in rat liver(27) . Western analysis with antibodies that rather specifically recognized the -isoform of PP-1 yielded a signal for both PP-1N and PP-1N (Fig. 6). The presence of the -isoform is corroborated by findings that the two holoenzymes could be immunoprecipitated with these antibodies (Fig. 3C). Surprisingly, the catalytic subunit of PP-1N migrated as a slightly larger protein than recombinant PP-1C and than the catalytic subunit of PP-1N. These differences may reflect a distinct posttranslational modification or the presence of two -isoforms generated, for example, by alternative splicing. Along the same line, it has been shown that there are two forms of PP-1 that only differ by the presence or absence of an amino-terminal 11-amino acid insert(16) .


Figure 6: Identification of the -isoform of the catalytic subunit in PP-1N. An aliquot of the Mono Q peak fractions of PP-1N and PP-1N, as well as 50 ng of each of the indicated recombinant isoforms of PP-1 were subjected to SDS-PAGE (10%) and blotted onto Immobilon membranes. Subsequently, the catalytic subunit was visualized with antibodies against PP-1, as explained under ``Experimental Procedures.''



Substrate Specificity

The above data imply that the noncatalytic subunit(s) of PP-1N and PP-1N 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 (1, 2, 3) , 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-1N increased the phosphatase activity 2-13-fold. On the other hand, a preincubation of PP-1N 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 1also 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.



Regulation of PP-1Nby Reversible Phosphorylation

We have previously shown that the active fragments of NIPP-1 from bovine thymus show a decreased affinity for PP-1 after phosphorylation by protein kinase A or casein kinase-2(11, 12) . In agreement with this result we found that after incubation of hepatic PP-1N 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-1 labeling was also obtained after phosphorylation of blotted NIPP-1 with PKA. Subsequent incubation with PP-2A and PP-2B restored binding of DIG-PP-1, 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-1N or blotted hepatic NIPP-1 with casein kinase-2 under phosphorylating conditions did not affect the extent of binding of DIG-PP-1 to NIPP-1 (not shown).

Although phosphorylation of native PP-1N by PKA decreased the affinity of NIPP-1 for DIG-PP-1, 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-1NR 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-1-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-1 to NIPP-1, with a maximal effect after 30-60 min. By 90 min the binding of DIG-PP-1 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-2A 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-1N or of the binding of DIG-PP-1 to R111 through phosphorylation by PKA or casein kinase-2.


DISCUSSION

PP-1N Is Controlled by Inhibitory Polypeptides

In two previous studies it was concluded that PP-1 is present in hepatic nuclear extracts as free catalytic subunit(7, 8) . 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 (7) and (8) , which presumably caused dissociation of the catalytic subunit. Moreover, at NaCl concentrations above 0.3 M, histones are extracted(27) , which have been shown to interfere with the phosphorylase phosphatase assay(1) . An additional explanation for the generation of free PP-1 in nuclear fractions is the extreme sensitivity of regulatory polypeptides to endogenous proteases ( Fig. 1and Fig. 2). Perhaps such proteolysis is mediated by the multicatalytic proteasome complex, which was shown to be present in liver nuclei(28) . In agreement with this proposal, we found that the degradation of the regulatory polypeptides of PP-1 is blocked by leupeptin ( Fig. 1and Fig. 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(29) .

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(1) . Such a strategy has for example recently been employed for the identification of the nuclear phosphatase(s) acting on p34-phosphorylated histone H1 (30) or PKA-phosphorylated cAMP response element binding protein, CREB(31) . 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 (1) ), 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-1 (Fig. 3). The same approach has previously been used for the identification of the PP-1-binding subunit of purified PP-1M(26) . It should be noted, however, that this approach does not necessarily allow one to identify all PP-1-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-1 after denaturing electrophoresis. Also, although it seems likely that the heat-stable inhibitor(s) of PP-1 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-1-labeling technique (not shown).

The Subunit Structure of PP-1N

The copurification and coimmunoprecipitation of NIPP-1 and PP-1N (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-1N is a heterodimer of PP-1 and NIPP-1. We suggest that PP-1N represents the native hepatic homolog of PP-1N, which was previously identified in thymus nuclear extracts(11, 12) . 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-1N (Fig. 3) suggest that R111 is a subunit of PP-1N. From gel filtration, we deduced an apparent molecular mass of about 160 kDa, which agrees with a heterodimeric structure between PP-1 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-1N and PP-1N contain the -isoform of the catalytic subunit (Fig. 6). Moreover, both species of PP-1N could be immunoprecipitated with PP-1-specific antibodies. However, since only about 70% of the activities of PP-1N and PP-1N were precipitated with these antibodies, it cannot be ruled out that a minor fraction of the holoenzymes contains other isoform(s) of PP-1.

Regulation of PP-1N

We found that the phosphorylation of NIPP-1 by PKA was associated with a lesser binding of PP-1 (Fig. 7). This agrees with similar findings on PP-1N from bovine thymus(11) . 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-1N 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-1N are independently controlled processes and that PKA only controls the reassociation of the holoenzyme.


FOOTNOTES

*
This work was supported by the ASLK, by the Belgian Fund for Medical Scientific Research (Grant 3.0041.93), and by a Concerted Research Action of the ``Vlaamse Executieve.'' The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
On leave from the Institute of Biochemistry and Biophysics (Polish Academy of Sciences) in Warsaw, Poland.

To whom correspondence should be addressed. Tel.: 32-16-345700; Fax: 32-16-345995.

The abbreviations used are: PP-1, protein phosphatase 1; PP-1, catalytic subunit of PP-1; NIPP-1, nuclear inhibitor of PP-1; PP-1N, nuclear PP-1; PP-1G, PP-1 associated with glycogen; PP-1M, PP-1 associated with myofibrils; PP-1E, PP-1 associated with endoplasmic reticulum; PP-1S, soluble fraction of PP-1; PP-2A, dimeric form of protein phosphatase-2A, previously called PCS or PP2A; TLCK, 1-chloro-3-tosylamido-7-amino-2-heptanone; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; DIG, digoxygenin; PAGE, polyacrylamide gel electrophoresis; PKA, protein kinase A.

A. Van Eynde, S. Wera, M. Beullens, S. Torrekens, F. Van Leuven, W. Stalmans, and M. Bollen, unpublished data.


ACKNOWLEDGEMENTS

We thank Dr. A. A. DePaoli-Roach for the generous gift of polyclonal antibodies against PP-1 and Dr. E. Y. C Lee for the recombinant isoforms of PP-1. The p34 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.


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