©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Interaction of the Ribosomal Protein, L5, with Protein Phosphatase Type 1 (*)

(Received for publication, March 14, 1995; and in revised form, May 30, 1995)

Katsuya Hirano (1) Masaaki Ito (2) David J. Hartshorne (1)(§)

From the  (1)Muscle Biology Group, Shantz Building, University of Arizona, Tucson, Arizona 85721 and the (2)First Department of Internal Medicine, Mie University, School of Medicine, Tsu, Mie 514, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The two-hybrid system was used to screen for binding proteins of type 1 protein phosphatase. Two plasmids were constructed, one containing the cDNA of the isoform of the type 1 catalytic subunit and the other containing a chicken gizzard cDNA library. Yeast (Y190) were transformed with the plasmids and screened for interacting species. 35 positive clones were categorized into 19 gene groups. Most of these were not identified. One clone, however, contained a sequence identical to the C-terminal portion of the chicken ribosomal protein L5 and corresponded to nucleotide residues 606-975. L5 was isolated from rat liver ribosomes as the L5bullet5 S RNA complex. This activated phosphatase activity of a myosin-bound phosphatase and the isolated type 1 catalytic subunit using phosphorylated myosin light chains and phosphorylase a as substrates. In addition, it was found that phosphatase sedimented with ribosomal subunits containing L5 but did not sediment with those deficient in L5. These data indicate that L5 binds to the catalytic subunit of the type 1 protein phosphatase and may act as a target molecule for phosphatase in ribosomal function or other cell mechanisms.


INTRODUCTION

Phosphorylation plays a vital role in many cellular processes. With respect to the Ser/Thr protein kinases, over 100 have been identified(1) , and this list is rapidly growing. In contrast, the number of protein phosphatases is more limited. The major classes of protein phosphatases directed to either phosphoserine or phosphothreonine are protein phosphatase type 1 (PP1), (^1)PP2A, PP2B, and PP2C(2) . Within each category, different isoforms exist for the catalytic subunits(3) . For the catalytic subunit of PP1, there are 4 isoforms: alpha, (1), (2), and (4) . In addition, the properties of the catalytic subunits may be modified by interaction with other subunits. This has been demonstrated with PP2A (5) where combinations of subunits are involved. These subunits may also function as targeting modules where the substrate and catalytic subunit are both bound to a common molecule. The targeting subunit provides proximity of enzyme and substrate and may have a regulatory function. The classical example is that of the glycogen- and sarcoplasmic reticulum-associated PP1 of skeletal muscle, termed PP1(G). The targeting molecule is the G subunit, and its properties are modified by phosphorylation at various sites(6, 7) .

Our interests are focused on the role of phosphorylation in smooth muscle function. Since it is evident that phosphatase subunits play a key role in the dephosphorylation mechanisms, it was decided to screen for PP1c-binding proteins in smooth muscle, namely gizzard. The two-hybrid system was used to isolate genes from a chicken gizzard cDNA library that encoded proteins that interact with PP1. The two-hybrid system was developed by Field and Song (8) and later modified(9, 10) . We used the modified system in which the combination of genetic selection for histidine prototrophy and the assay for beta-galactosidase activity is applied to identify positive clones. This system reduces the possibility of false positives(9, 10) . Screening of the gizzard library identified 35 positive clones that were grouped into 19 gene groups. Most of the nucleotide or derived amino acid sequences from these clones were not identified and did not match known sequences. Two of the clones were very similar to known proteins, the human splicing factor and the chicken ribosomal protein L5. This study investigates L5, and it is suggested based on the following data that L5 is a PP1c-binding protein.


EXPERIMENTAL PROCEDURES

Materials

Oligonucleotides were synthesized at the Macromolecular Structure Facility at the University of Arizona. Taq DNA polymerases were from Perkin-Elmer Corp. or Boehringer Mannheim. Other enzymes were from Boehringer Mannheim, Promega, or Pharmacia Biotech Inc. Media for bacterial and yeast cultures were from Difco. Radionucleotides ([-P]ATP, [alpha-S]dATP) were from DuPont NEN. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside was from Research Products International. The anti-PP1 polyclonal antibody was a generous gift from Dr. M. Nagao (National Cancer Center Research Institute, Tokyo). The anti-hemagglutinin (anti-HA) antibody was from Berkley Antibody.

Bacterial and Yeast Strain and Media

Escherichia coli DH5alpha was used for transformation recipient for all plasmid constructions. E. coli XL1-Blue MRF` was used as a host strain for phage Uni-ZAP XR (Stratagene). E. coli M15[pREP4] (Qiagen) was used for expression of recombinant phosphatase type-1 (rPP1). Saccharomyces cerevisiae Y190 was used for screening chicken gizzard cDNA library for clones interacting with PP1 (donated by Dr. S. J. Elledge, Baylor College of Medicine, Houston, TX)(10) . Media were prepared as described(11, 12) .

Construction of Bait Plasmids for the Two-hybrid System

The full-length cDNA of human aortic PP1 (327 amino acids) was obtained by PCR-mediated amplification from a single plaque of gt10(13) . The primers were designed to introduce NcoI and BamHI sites for cloning (5`-GACACCATGGCGGACGGGGAG-3` for sense primer and 5`-TTGGATCCTTCACCTTTTCTTCGGCGG-3` for antisense primer). The PCR product showed an apparent single band of the expected size (999 base pairs) on agarose gel. The PCR product was digested with NcoI and BamHI, purified on 1% agarose gel, and ligated to the expression plasmid, pAS1 (donated by Dr. S. J. Elledge)(9) . E. coli DH5alpha was transformed and selected for ampicillin resistance. This construct encodes a fusion protein of GAL4 DNA binding domain (1-147 amino acids), influenza viral hemagglutinin (HA), epitope-containing peptides (20 amino acids), and the full-length of PP1(9) .

Chicken gizzard myosin light chain kinase and the inverted sequence of PP1 were used as control baits. NdeI-SmaI fragment of pET.E972 (14) was ligated to the NdeI and SmaI site of pAS1. The construct (pAS1-MK) encoded the C-terminal half of the enzyme (from Leu-447 through Glu-972) tagged by the GAL4 DNA binding domain. The full-length cDNA of PP1 was enzymatically amplified as described above except that primers were designed to introduce a BamHI site at the 5`-end and a NcoI site at the 3`-end (5`-GGGGGGATCCAGATGGCGGACGGGGAG-3` for sense primer and 5`-GAATTCCATGGTTCACCTTTTCTTCGG-3` for antisense primer). The BamHI and NcoI-digested PCR product was ligated into the NcoI and BamHI sites of pAS1, resulting in an inverted insertion of PP1 cDNA. This construct (pAS1-iPP1) encoded a 37-mer peptide deduced from the inverted PP1 cDNA after HA tag.

Construction of Chicken Gizzard cDNA Library

Poly(A) RNA was isolated from fresh chicken gizzard using an oligo(dT) column (Fast Track kit, Invitrogen). cDNA was synthesized using both oligo(dT) and random hexamer primers for the first strand synthesis. The cDNA library was constructed in Uni-Zap phage (Stratagene), where the cDNA library was inserted unidirectionally in EcoRI and XhoI sites of the phage chromosome. Phage DNA was isolated from plate lysate, and the inserts of the library were amplified by PCR with SK (5`-CGCTCTAGAACTAGTGGATC-3`) and M13-20 (5`-GTAAAACGACGGCCAGT-3`) primers. A plasmid pGAD424 (Clontech) digested with EcoRI and SalI was purified using sucrose density gradient (10-30%) centrifugation(15) . This procedure removed trace amounts of uncut plasmid and was necessary to obtain high efficiency and a high recombinant ratio in transformation. EcoRI and XhoI-digested inserts of the library were ligated with the pGAD424 and used to transform E. coli DH5alpha. Ampicillin-resistant colonies were pooled. Totally, the ligation of approximately 0.5 µg of library cDNA and 1 µg of plasmid resulted in a cDNA library with 4 10^6 independent clones.

Two-hybrid Screening of the Chicken Gizzard cDNA Library

S. cerevisiae Y190 was transformed with pAS1-PP1 by the lithium acetate method(16) . Yeast cells containing pAS1-PP1 (Y190/pAS1-PP1) were selected on synthetic complete media lacking tryptophan (SC-Trp). Y190/pAS1-PP1 cells were transformed with the chicken gizzard cDNA library constructed in pGAD424. Transformed cells were plated on SC-Try-Leu-His containing 30 mM 3-aminotriazole (9) and incubated for 10 days. His colonies were screened for beta-galactosidase activity using a filter lift assay (17) and the chromogenic substrate 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside. Positive colonies were cloned by repeating the plating of the positive colonies and assays for beta-galactosidase (at least twice).

Recovery of Library Plasmid from Yeast Cells

Yeast nucleic acid was prepared from positive clones as described (18) except that the aqueous phase was further extracted with chloroform/isoamyl alcohol (24:1), and the nucleic acid was precipitated in ethanol and dissolved in 10 mM Tris-HCl, 1 mM EDTA, pH 8.0. This yeast nucleic acid preparation contained both pAS1-PP1 and pGAD424 library cDNA. E. coli DH5alpha was transformed with the yeast nucleic acid and selected for ampicillin resistance. Plasmid DNA was prepared from several ampicillin-resistant colonies and examined for its restriction pattern. Bacterial colonies showing the restriction pattern characteristic of pGAD424 were used for further investigation.

DNA Sequence Analysis

DNA sequences were determined by the dideoxy-mediated chain termination method (19) with [alpha-S]dATP and Sequenase DNA polymerase version 2.0 (U. S. Biochemical Corp.). Sequence analysis and homology searches were performed using the GCG package (Genetic Computer Group).

Expression of PP1 as a Hexahistidine-tagged Protein

The full-length human aortic PP1 cDNA was amplified using PCR with primers designed to introduce BamHI and PstI sites for subcloning (5`-GGGGGGATCCAGATGGCGGACGGGAG-3` for sense primer and 5`-GGGCTGCAGTTCACCTTTTCTTCGGCGG-3` for antisense primer). The PCR product was digested with BamHI and PstI, purified on agarose gel, and ligated to plasmid pQE32 digested with BamHI and PstI (Qiagen). This construct encoded hexahistidine-tagged PP1. The 13-mer peptide, Met-Arg-Gly-Ser-His-His-His-His-His-His-Gly-Ile-Gln, was added to the N terminus of PP1. The expression plasmid, pQE-PP1, was introduced into the E. coli host strain, M15[pREP4] (Qiagen). Expression was induced by 0.4 mM isopropyl-beta-D-thiogalactopyranoside. The cells were collected, and the pellet was homogenized in 6 M guanidine HCl, 0.1 M sodium phosphate, 10 mM Tris-HCl, pH 8.0 (buffer A). The supernatant obtained by centrifugation at 10,000 g for 15 min, 4 °C, was mixed with nickel-nitrilotriacetic acid-agarose resin equilibrated with Buffer A at room temperature for 45 min. The resin was loaded onto a column and washed with buffer A and then with 6 M guanidine HCl, 0.1 M sodium phosphate, 10 mM Tris-HCl, pH 6.3 (buffer B). rPP1 bound to the resin via the hexahistidine tag was eluted with 0.25 M imidazole in buffer B. The eluate was rapidly diluted 25-100-fold in 30 mM Tris-HCl, 0.8 M NaCl, 0.02% Tween 20, 1 mM MnCl(2), 10 mM dithiothreitol, 10 µg/ml leupeptin, 10 µM 4-amidinophenylmethanesulfonyl fluoride, pH 7.0, and stirred at 4 °C for 6-7 h(20, 21) . The diluted sample was dialyzed against 30 mM Tris-HCl, 30 mM NaCl, 1 mM MnCl(2), 1 mM dithiothreitol, pH 7.0. The dialysate was clarified by centrifugation at 27,000 g for 10 min, 4 °C, and the supernatant was concentrated using centriprep-10 microconcentrators (Amicon).

Preparation of 80 S Ribosome, EDTA-treated Large Subunit, and L5

Initially, the expression of L5 and the C-terminal part of L5 encoded by clone 25 was attempted in E. coli, using the pQE expression vector. The C-terminal fragment was expressed, but expression of L5 proved lethal, and it was necessary to isolate L5 from animal tissue. The rat liver 80 S ribosome was isolated(22) , treated with EDTA, and fractionated by sucrose density gradient (10-30%) centrifugation(23, 24) . There were three peaks. The peak at lowest density showed a single band on SDS-PAGE with a mobility similar to L5 (i.e. 38 kDa). This peak, containing the L5bullet5 S RNA complex, was dialyzed against 20 mM Tris-HCl, 100 mM KCl, 2 mM MgCl(2), pH 7.5 (L5 buffer) and concentrated using centriprep-10 (Amicon). The concentration of the L5bullet5 S RNA complex thus obtained was estimated to be 2.5 µM. The peak at highest density was also pooled, dialyzed against the same buffer, and concentrated. This peak represented the large subunit lacking the L5bullet5 S RNA.

Assay for Phosphatase Activity

P-Labeled myosin light chain (25) and phosphorylase a(26) were prepared as substrates. Phosphatase activity was measured with 5 µMP-labeled myosin light chain or 3 µMP-labeled phosphorylase a in L5 buffer with or without 0.3 mM CoCl(2). The reaction was started by addition of substrates and terminated by addition of trichloroacetic acid and bovine serum albumin to final concentrations of 7% and 4 mg/ml, respectively. The precipitated protein was sedimented, and radioactivity of the supernatant was determined by Cerenkov counting.

Coprecipitation of Phosphatase with Ribosomal Fractions

Ribosomal fractions (80 S ribosome or EDTA-treated large subunit) were mixed at 0.4 µM with phosphatase in L5 buffer containing 0.3 mM CoCl(2). The mixture was centrifuged at 170,000 g for 1 h at 25 °C with an air-driven ultracentrifuge (Beckman Airfuge). A parallel mixture was not centrifuged. The supernatant and non-centrifuged mixture were assayed for phosphatase activity in L5 buffer containing 0.3 mM CoCl(2) with the two substrates, as described above.

Other Preparations

The myosin-bound phosphatase (27) , myosin light chain(28) , and myosin light chain kinase (29) were prepared from turkey gizzard. Calmodulin was prepared from bovine testis(29) . The catalytic subunit of PP1 was prepared from rabbit skeletal muscle(30) .

Other Methods

Protein concentrations were determined by the BCA (Pierce) or Bradford (Bio-Rad) methods with bovine serum albumin as standard. SDS-PAGE(31) , Western blot techniques(32) , and standard manipulation of nucleic acids(11) , bacteria, and yeast (11, 33) were as described.


RESULTS

Isolation of Protein Phosphatase Type 1-interacting Proteins

S. cerevisiae Y190 was transformed with a plasmid (pAS1-PP1) that encodes the DNA-binding domain-tagged PP1. Fig. 1verifies the expression of a fusion protein containing the DNA-binding domain of GAL4 and PP1. Western blots with anti-PP1 antibody revealed that only the yeast cell homogenate transformed with pAS1-PP1 (lane7) showed a positive band of the expected size (59 kDa). Homogenates of non-transformed cells or cells transformed with vector plasmid alone were not reactive. A band of appropriate mass also was recognized by the anti-HA antibody, again only in the homogenate of the pAS1-PP1-transformed cells (lane3). This antibody cross-reacted with a band of low molecular mass (18 kDa) in the homogenate of cells transformed with vector alone (lane2). This band probably represented the fusion product of the GAL4 DNA-binding domain and the HA tag. These findings indicate that a whole fusion protein was expressed correctly (in-frame) from the GAL4 DNA-binding domain through the HA epitope to the C terminus of PP1 in Y190/pAS1-PP1 cells. beta-Galactosidase activity was not detected in Y190/pAS1-PP1. The expression level of the PP1-fusion protein was estimated by Western blot analysis using rPP1 expressed in E. coli as a standard. The Y190/pAS1-PP1 cells were cultured on a SC-Trp-Leu plate for 3 days. Several colonies were selected and cultured in liquid medium overnight. Cellular pellets obtained after the overnight culture were applied to SDS-PAGE. The concentration of the GAL4-PP1 fusion protein in yeast was estimated to be 40 ± 20 nM (n = 9).


Figure 1: Western blots of yeast cell homogenates with anti-hemagglutinin and anti-PP1 polyclonal (rabbit) antibodies. Homogenates of non-transformed (lanes1 and 5), vector-transformed (lanes2 and 6), and pAS1-PP1-transformed cells (lanes3 and 7) were separated on 12.5% gels, transferred, and probed with anti-HA (lanes1-3) and anti-PP1 (lanes4-7). Lane4 is the myosin-bound phosphatase from gizzard. The enhanced chemiluminescence method was used to detect the peroxidase-conjugated anti-rabbit IgG antibodies. The singlearrowhead indicates the 59-kDa fusion protein recognized by both antibodies. The doublearrowhead indicates the 38-kDa PP1 catalytic subunit. The arrow shows the 18-kDa band recognized by the anti-HA antibody. Molecular mass markers are shown on the left.



To screen a chicken gizzard cDNA library, the Y190/pAS1-PP1 cell was transformed with the library plasmid, selected for His, and assayed for beta-galactosidase activity. After screening the library twice, a total of 35 colonies of His and beta-galactosidase were obtained. All colonies were cloned by repetitive plating on SC-Trp-Leu agar and assayed for beta-galactosidase. Library plasmids were recovered in E. coli DH5alpha (see ``Experimental Procedures''). The patterns of digestion by EcoRI and PstI were examined for all library plasmids, and partial or full-length DNA sequences of inserts of all clones were determined. 35 positive clones were categorized into 19 gene groups, since some clones had the same insert. Most of these genes were not identified by nucleotide or deduced amino acid similarities to sequences in the data base. However, five genes were matched to known sequences. Two were mitochondrial proteins but were probably prematurely terminated. The other 3 genes encoded proteins identical or similar to the human splicing factor (clone 24), the regulatory subunit of the glycogen-bound phosphatase (clone 3), and the chicken ribosomal protein, L5 (clone 25). This study focuses on the characterization of L5 as a phosphatase-binding protein.

Characterization of L5

The full nucleotide sequence of clone 25 was determined from both strands and is shown in Fig. 2. It contained 384 base pairs and a poly(A) tail. All of the determined sequences except for the adaptor sequences were found to be identical to the chicken ribosomal protein L5 (residues 606-975) as reported by Kenmochi et al.(34) . The deduced amino acid sequence is identical to the C-terminal portion of L5 (residues 202-297).


Figure 2: The nucleotide and derived amino acid sequences of clone 25 compared to chicken L5. The DNA sequence of clone 25 is shown from the 5`-ligation site. The deduced amino acid sequence of clone 25 is in-frame continuing from the GAL4 activation domain. Nucleotide and amino acid numbering for L5 is as previously described(37) . Asterisk indicates termination codon. The underlinedsequence at the 5`-end is from an adaptor used in the construction of the Uni-ZAP cDNA library, and the underlinedsequence at the 3`-end is the sequence from a vector downstream of the cDNA insertion site.



Before additional studies were carried out, it was essential to determine if clone 25 was a false positive. For this purpose, we constructed control bait plasmids as described under ``Experimental Procedures.'' The following results indicate that clone 25 is not a false positive. First, clone 25 alone did not activate beta-galactosidase activity in Y190 cell (data not shown). Second, Y190 cells were transformed with either pAS1-PP1, pAS1-MK, or pAS1-iPP1. Expression of fusion proteins in each transformed cell was verified by Western blot analysis using anti-HA antibody, anti-PP1 antibody, or anti-myosin light chain kinase antibody(21) . All of the transformed cells had no detectable beta-galactosidase activity. Next, the cells were transformed with the pGAD424-clone 25. Only those cells containing pAS1-PP1 showed beta-galactosidase activity after introduction of clone 25.

Activation of Phosphatase Activity by L5

The results from the two-hybrid screening suggest that L5 is a binding protein of PP1. Binding could influence phosphatase activity, and thus any alteration of activity by L5 would be an additional indication of the interaction between PP1 and L5. Results shown in Fig. 3suggest that L5 is an activator of PP1 activity. L5 was isolated as the L5bullet5 S RNA complex from rat liver ribosomes by the EDTA treatment. Phosphatases used were the turkey gizzard MBP, the catalytic subunit of PP1 from rabbit skeletal muscle (PP1c), and the rPP1 expressed as a hexahistidine-tagged protein. When P-labeled myosin light chain was used as substrate, L5 increased phosphatase activity of MBP in the presence of 0.3 mM CoCl(2). Activation was observed at 8 nM L5 and was maximum (approximately 2.5-fold activation) at 150 nM (Fig. 3A). With P-labeled phosphorylase a as substrate, activation of MBP by L5 also was observed, although the extent of activation was less than with light chains as substrate (Fig. 3B). MBP activity is Co dependent(35) , and activation by L5 in the absence of Co with either substrate was not observed (data not shown). The activity of PP1c also was activated by L5 using either substrate (Fig. 3, A and B). The extent of activation was less than MBP with light chains as substrate but slightly more than MBP with phosphorylase a. The isolated PP1c is not markedly Co dependent, but it is interesting that activation by L5 was only observed in the presence of Co. The activity of rPP1 was not activated by L5 for either substrate in the presence or absence of Co (absence of Co shown in Fig. 3, A and B). The basal activity of rPP1 with either substrate was inhibited by 0.3 mM CoCl(2).


Figure 3: Effect of rat liver L5bullet5 S RNA on phosphatase activity using P-labeled 20-kDa light chains (A) or phosphorylase a (B). Activity is expressed as a percent of control activity. Myosin-bound phosphatase (n = 5) plus 0.3 mM CoCl(2), black square; catalytic subunit from rabbit skeletal muscle (n = 3) plus (bullet) and minus CoCl(2) (); recombinant PP1 (n = 3) minus CoCl(2) (). Data are mean ± S.E.



Kinetics of activation of MBP by L5 using phosphorylated light chains as substrate are shown in Fig. 4. V(max) was increased from 0.65 ± 0.33 µmol/min mg protein (n = 3) to 1.14 ± 0.49 µmol/min mg protein (n = 3) by 500 nM L5. K values (4.4 ± 2.0 and 3.7 ± 1.4 µM in the absence and presence of L5, respectively) were unchanged.


Figure 4: Effect of rat liver L5 on phosphatase activity. Data obtained with myosin-bound phosphatase and phosphorylated light chain (LC) plus 0.3 mM CoCl(2) in the presence (bullet) and absence () of 0.5 µM L5bullet5 S RNA complex. Data are mean of three assays. V(max) and K values were obtained by linear regression.



Since L5 was isolated as the L5bullet5 S RNA complex, it is possible that RNA may be involved in the activation of phosphatase activity. To test this possibility, the L5bullet5 S RNA complex was treated with 20 µg/ml RNaseA for 1 h at 30 °C. Urea-polyacrylamide gel electrophoresis (7 M urea, 6% acrylamide) revealed the disappearance of RNA in the RNaseA-treated complex (data not shown). The effect of the RNaseA-treated L5 was identical to non-treated L5, as judged from the concentration dependence of activation (data not shown). It is likely, therefore, that activation of phosphatase activity is due to the protein component of the L5 complex.

Binding of Phosphatase to Ribosomes

The binding of MBP to ribosomes was examined using a sedimentation assay (``Experimental Procedures''). The 80 S ribosome and the EDTA-treated ribosomal fraction were used. SDS-PAGE showed that approximately 95% of the ribosomal preparations were sedimented under the conditions used. In the presence of the 80 S ribosome (0.4 µM), the activity of MBP (2 µg/ml) was 181 ± 11% (using light chain as substrate) and 131 ± 11% (using phosphorylase a as substrate). After centrifugation, the percent activity remaining in the supernatant was 25 ± 9 and 37 ± 11% for the two substrates, respectively (Fig. 5). For the EDTA-treated 80 S fraction, the activities before centrifugation were 155 ± 23% (light chain) and 219 ± 33% (phosphorylase a). After centrifugation, the activities in the supernatant were not decreased (Fig. 5) and were 167 ± 14 and 237 ± 27%, respectively. Since the EDTA treatment removes L5 from the 80 S ribosome, these data add support to the idea that L5 is a phosphatase-binding protein. In preliminary experiments, it was found that rPP1 also bound to the 80 S ribosome (in the absence of Co) but did not bind to the EDTA-treated fraction.


Figure 5: Cosedimentation of phosphatase activity with ribosomal fractions. Myosin-bound phosphatase was incubated with 0.4 µM 80 S ribosome (A and C) or 0.4 µM EDTA-treated large ribosomal subunit (B and D). Phosphatase activities of the mixture before centrifugation (solidbars) and supernatant after centrifugation (openbars) were measured using light chains (A and B) or phosphorylase a (C and D) as substrates. Activity is expressed as percent of control minus the ribosomal fraction. Data are means ± S.E. (n = 3).




DISCUSSION

The major finding of this study is that the ribosomal protein, L5, is a binding protein for the PP1 catalytic subunit. This assertion is supported by the following three lines of evidence: 1) the strongest evidence is that L5 was selected as an interacting species with PP1 by the two-hybrid system; 2) the multisubunit MBP and the isolated PP1c were activated by L5, indicating an interaction; and 3) the MBP was cosedimented with ribosomes containing L5 but not with a ribosomal fraction depleted of L5.

Thus, one function of L5 might be to target PP1c to the L5 site on the ribosome. Whether the activation of phosphatase activity also is an in vivo function of L5 is not known. The activation of phosphatase activity, observed in this study, was Co dependent. Although activation by Co or Mn is found frequently (see Refs. 35, 36), it is difficult to rationalize its physiological significance. It may mimic a cellular mechanism, but such is not identified. Since rPP1 was not activated by L5 nor was Co dependent, it is possible that some post-translational modification of the catalytic subunit is required to link activation by L5 and Co dependence.

The location of the L5-binding site on PP1 is not known. However, it is suggested that this is not in the C-terminal region of PP1. The PP1c isolated from animal tissues is usually obtained in a truncated form (35 kDa) lacking the C-terminal 30-40 residues(37) . This is indicated in our study since the 35-kDa PP1c did not react with the PP1 antibody raised against the C-terminal sequence(38) . The activity of PP1c was activated by L5, and thus it is unlikely that the C-terminal region of PP1c is involved in the binding site. There is some difference in the N-terminal sequences of the 4 PP1c isoforms(4) , but the central core of about 250 residues is highly conserved. Thus, if the binding site for L5 is within the central core, all of the PP1c isoforms might be expected to bind to L5.

The L5bullet5 S RNA complex has several interesting features. The complex is found in the nucleolus, the cytoplasm, and nucleoplasm as well as being a component of the large ribosomal subunit (60 S). Only half of the 5 S RNA in mammalian cells is associated with the 60 S ribosome (39) , and a substantial fraction of the non-ribosomal 5 S RNA is bound to L5(40) . Thus, the physiological role of the L5-phosphatase interaction could be a part of ribosomal function or associated with the non-ribosomal L5bullet5 S RNA complex.

Cross-linking studies with rat liver ribosomes (41) revealed that L5 is located at the interface between the large and small subunits, which is the site of protein synthesis. Several proteins of the large and small subunits were proximal including S6. In another study, it was found that S6, S3a, L5, and L6 were cross-linked to mRNA(42) . S6 is known to be phosphorylated in vivo(43) and is phosphorylated in response to several factors that stimulate protein synthesis (for reviews, see (44) and (45) ). PP1 is the major phosphatase for S6 (2) in several cells. Thus, L5 could target PP1 to the ribosomal subunit interface for the purpose of dephosphorylation of S6. In this scenario, PP1 would act as a negative regulator of protein synthesis.

The function of the nuclear and cytosolic L5bullet5 S RNA (non-ribosomal) is not established. Some interesting possibilities exist. It was reported that L5 and p53 were coprecipitated by mdm-2-specific antibodies(46) , and it is known that p53 is regulated by phosphorylation(47) . The L5-bound phosphatase could dephosphorylate p53 in the p53-mdm-2-L5-phosphatase complex. In addition, it is known that PP1 is present in the nuclear chromatin/matrix fraction(37) . The translocation of phosphatase between particulate and cytosolic fractions has been suggested(48) , and it is possible that the L5bullet5 S RNA-PP1 complex acts as a nuclear transporter, or shuttle, for PP1. A cytosolic L5bullet5 S RNA complex was copurified with the aminoacyl-tRNA synthetase complexes from rat liver(49) , and it was shown that the L5 complex activated synthetase activity 2-3-fold(49, 50) . However, it is not known whether tRNA synthetase activity is regulated by phosphorylation.

The above results have shown that one of the PP1-binding proteins in chicken gizzard is the ribosomal protein, L5. Rat ribosomal L5 was prepared, and this also binds PP1. The two proteins are 96% identical in amino acid sequence. The functional implications of the L5-PP1 interaction are not known. However, since L5 could act as a target molecule for PP1, this raises the possibility that the functions of the two proteins are coordinated.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants HL 23615 and HL 20984 (to D. J. H.) and by grants-in-aid for scientific research and for scientific research on priority areas from the Ministry of Education, Science, and Culture of Japan (to M. I.). 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.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].

§
To whom correspondence should be addressed. Tel.: 520-621-7239; Fax: 520-621-1396.

(^1)
The abbreviations used are: PP1, PP2A, PP2B, and PP2C, protein phosphatases type 1, type 2A, type 2B, and type 2C, respectively; PP1alpha, PP1(1), PP1(2), and PP1, four isoforms of the catalytic subunit of PP1; PP1c, catalytic subunit of PP1; rPP1, recombinant PP1; MBP, myosin-bound phosphatase; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; HA, hemagglutinin.


ACKNOWLEDGEMENTS

We are indebted to Dr. D. Bourque (Biochemistry Dept., University of Arizona) for encouragement and advice with the ribosomal preparations.


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