(Received for publication, March 14, 1995; and in revised form, May 30, 1995)
From the
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 L5
5 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.
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), ()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:
,
,
, 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
. 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
-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.
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.
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
-galactosidase
activity. After screening the library twice, a total of 35 colonies of
His
and
-galactosidase
were
obtained. All colonies were cloned by repetitive plating on SC-Trp-Leu
agar and assayed for
-galactosidase
. Library
plasmids were recovered in E. coli DH5
(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.
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 -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
-galactosidase activity. Next, the cells were
transformed with the pGAD424-clone 25. Only those cells containing
pAS1-PP1
showed
-galactosidase activity after introduction of
clone 25.
Figure 3:
Effect
of rat liver L55 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
,
;
catalytic subunit from rabbit skeletal muscle (n = 3)
plus (
) and minus CoCl
(
); recombinant PP1
(n = 3) minus CoCl
(
). Data are mean
± S.E.
Kinetics of activation of MBP by L5 using phosphorylated light
chains as substrate are shown in Fig. 4. V 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 in the presence (
) and absence (
) of
0.5 µM L5
5 S RNA complex. Data are mean of three
assays. V
and K
values were obtained by linear
regression.
Since L5 was isolated
as the L55 S RNA complex, it is possible that RNA may be involved
in the activation of phosphatase activity. To test this possibility,
the L5
5 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.
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).
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 L55 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 L5
5 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 L55 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
L5
5 S RNA-PP1 complex acts as a nuclear transporter, or shuttle,
for PP1. A cytosolic L5
5 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank[GenBank].