(Received for publication, July 10, 1995; and in revised form, August 30, 1995)
From the
NIPP-1 was originally isolated as a potent and specific nuclear
inhibitory polypeptide (16-18 kDa) of protein phosphatase-1. We
report here the cDNA cloning of NIPP-1 from bovine thymus and show that
the native polypeptide consists of 351 residues and has a calculated
mass of 38.5 kDa. The bacterially expressed central third of NIPP-1
completely inhibited the type-1 catalytic subunit, but displayed a
reduced inhibitory potency after phosphorylation by protein kinase A
and casein kinase 2. Translation of NIPP-1 mRNA in reticulocyte lysates
resulted in the accumulation of both intact NIPP-1 and a smaller
polypeptide generated by alternative initiation at the codon
corresponding to Met. A data base search showed that the
COOH terminus of NIPP-1 is nearly identical to the human ard-1 protein
(13 kDa), which has been implicated in RNA processing (Wang, M., and
Cohen, S. N.(1994) Proc. Natl. Acad. Sci. U. S. A. 91,
10591-10595). Comparison of the cDNAs encoding ard-1 and NIPP-1
suggests that their mRNAs are generated by alternative splicing of the
same pre-mRNA. Western blotting with antibodies against the COOH
terminus of NIPP-1, however, showed a single polypeptide of 47 kDa,
which was enriched in the nucleus. Northern analysis revealed a single
transcript of 2.2 kilobases in bovine thymus and of 2.4 kilobases in
various human tissues.
The serine/threonine protein phosphatases of type-1 (PP-1) ()comprise an abundant and phylogenetically conserved group
of enzymes that dephosphorylate key proteins in various cell
processes(1, 2) . All known species of PP-1 possess an
isoform of the same catalytic subunit (PP-1
), which
accounts for their specific inhibition by polypeptides like
inhibitor-1, inhibitor-2, and NIPP-1. The activity, substrate
specificity, and subcellular localization of type-1 protein
phosphatases is largely determined by their noncatalytic subunits.
PP-1 is more abundant in the nucleus (PP-1N) than in other cell
compartments. Although the nuclear holoenzyme(s) have not yet been
fully characterized, it has been established that the nucleus contains
various regulatory polypeptides of PP-1. Thus, it has been demonstrated
that inhibitor-2, which is abundant in the cytoplasm, is also partially
nuclear during the S-phase of the cell cycle(3) . In Schizosaccharomyces pombe a polypeptide has been identified,
designated sds22, that is enriched in the nucleus and is associated
with PP-1(4) . Durfee et al.(5) demonstrated an association of the type-1 catalytic subunit
with the retinoblastoma protein during mitosis and early G
.
We have isolated polypeptides of 16-18 kDa from bovine thymus,
termed NIPP-1, that are extremely potent and specific inhibitors of
PP-1(6) . Recent data suggest that the isolated inhibitors are
generated from a 41-kDa polypeptide by proteolysis(7) . In
nuclear extracts native NIPP-1 is present as an inactive heterodimeric
complex with PP-1
, designated PP-1N
.
Phosphorylation of NIPP-1 in PP-1N
by protein kinase A,
both in vitro and in vivo, does not cause the
dissociation or activation of the phosphatase but prevents the
reassociation of PP-1
with NIPP-1, following the
dissociation of the complex. In contrast, phosphorylation of the
16-18-kDa fragments of NIPP-1 by protein kinase A or casein
kinase 2 causes the release of active catalytic
subunit(8, 9) .
We report here the cDNA cloning of NIPP-1 from bovine thymus and show that the inhibitory region is localized in the central third of the polypeptide, which also contains multiple phosphorylation sites for protein kinase A and casein kinase 2. Native NIPP-1 is a protein with a calculated mass of 38.5 kDa, but initial evidence suggests that fragments of NIPP-1 may also be generated by alternative initiation of translation and by alternative RNA splicing.
Figure 1:
Microheterogeneity of NIPP-1 as
revealed by twodimensional electrophoresis. NIPP-1 purified from bovine
thymus nuclei until after reversed phase chromatography (6, 8) was P-labeled by phosphorylation
with protein kinase A, subjected to two-dimensional electrophoresis,
and visualized by autoradiography. All seven spots marked by an arrowhead represent species of NIPP-1, as indicated by
activity assays after blotting, elution, and dephosphorylation of the
individual species (not shown).
Western analysis of
a cytosolic fraction and a nuclear salt extract from rat liver was
essentially done as in (7) , with the affinity-purified NIPP-1
antibodies at a final concentration of 2 µg/ml. The nuclear salt
extract was prepared as described in (7) . The cytosolic
fraction was obtained by successive centrifugations at 8,000 g (10 min) and 150,000
g (35 min) of an
aliquot of the same homogenate that was also used for the preparation
of liver nuclei.
Nucleotide sequencing on both strands was performed using the dideoxynucleotide chain termination method (16) in an automated laser fluorescent DNA sequencer. Both vector-specific and cDNA-specific oligonucleotide primers were used.
The pBl-2175 and pBl-657 plasmids were
linearized near the 3`-end of the insert by digestion with XhoI and used as template for in vitro transcription
starting from the T3 promoter. The resulting capped RNA transcripts
were quantified by ethidium bromide staining on agarose gels and
translated in a rabbit reticulocyte lysate during 60 min at 30 °C
in the presence of 14 µM [S]methionine (14 mCi/ml). The translation
products were subjected to 10% Tricine-SDS-PAGE and visualized by
autoradiography.
Figure 2:
Nucleotide and predicted amino acid
sequence of NIPP-1 from calf thymus. In A the nucleotide
sequence is presented in the 5` to 3` direction and is numbered on the left. The deduced protein sequence is given below the
nucleotide sequence (one-letter code) and is numbered on the right. Also indicated are the three consensus initiator codons (bold), the stop codon (asterisk), the three putative
polyadenylation signals (boxed), and the primary structure
that was also obtained from NH-terminal sequencing of
peptides obtained after limited proteolysis (underlined). B schematically illustrates some basic features of the deduced
primary structure of NIPP-1, represented by the bar. Indicated
are potential nuclear localization signals (NLS), putative
phosphorylation sites for protein kinase A (PKA) and casein
kinase 2 (CK2), the occurrence of basic and acidic domains,
and the pI of some of these domains.
Using the first ATG
as start codon, pBl-2175 showed an open reading frame of 1053 bp,
encoding a polypeptide of 351 residues with a calculated mass of 38.5
kDa (Fig. 2A). Both sequences obtained from peptide
analysis were present in the central part of the predicted primary
structure, except for the residue corresponding to Ser,
which did not yield any signal during peptide sequencing. A likely
explanation for this discrepancy is that Ser
was
phosphorylated in the sequenced peptide fragment, which prevents
detection by standard gas-phase and pulse-liquid sequencing
procedures(20) . The 5-kDa peptide that contained Ser
was indeed radioactively labeled. Moreover, the basic residues
NH
-terminal to Ser
make it a perfect site for
phosphorylation by protein kinase A, which we used to phosphorylate
NIPP-1 prior to proteolysis.
Analysis of the deduced amino acid
sequence of NIPP-1 shows that 10% of the residues are proline, which is
twice above average and suggests a relatively low abundance of
-helices and
-sheets. Although NIPP-1 is a neutral
polypeptide (pI = 7.4), the charge is distributed unevenly (Fig. 2B). Indeed, while the NH
-terminal
and COOH-terminal thirds of NIPP-1 are rather basic, the central third
of the polypeptide is very acidic. Consistent with the nuclear
localization of NIPP-1, three putative nuclear localization signals are
present (Fig. 2B). As expected from our previous work
on purified NIPP-1(8, 9) , the primary structure also
shows several putative phosphorylation sites for protein kinase A
(Ser
, Ser
, Thr
) and casein
kinase 2 (Thr
, Thr
, Thr
,
Thr
, Ser
, Thr
), which are
largely clustered in the central acidic domain (Fig. 2B).
Figure 3: Schematic comparison of the mRNAs encoding NIPP-1 and ard-1. The large bars represent the mRNAs encoding bovine NIPP-1 (2175 bp) and human ard-1 (2401 bp). The open, numbered bars represent fragments that have more than 90% sequence identity. The hatched boxes represent domains that are specific for the mRNAs encoding NIPP-1 or ard-1. The small solid bars delineate the coding regions.
Using the BLAST program(21) , we also found 40% identity of two fragments (residues 156-195 and 232-253) of NIPP-1 with domains of the heavy chain of rabbit myosin. Furthermore, residues 253-289 of NIPP-1 were 31% identical to a domain in the 70-kDa RNA-binding protein of the U1 small nuclear ribonucleoprotein complex.
Figure 4:
In vitro translation of NIPP-1.
mRNAs encoding full-length NIPP-1 or a NIPP-1 fragment (residues
143-351) were prepared by in vitro transcription from
pBl-2175 or pBl-657, respectively. In vitro translation was
performed in reticulocyte lysates in the presence of
[S]methionine. After translation the lysates
were either directly boiled in SDS-sample buffer or first boiled (5
min) and centrifuged (1 min at 10,000
g) before
addition of the heat-stable fraction to the SDS-sample buffer.
Following 10% Tricine-SDS-PAGE the translation products were visualized
by autoradiography. Lanes 1 and 2 show the
translation products of the full-length NIPP-1 mRNA in total lysates
and in the heat-stable lysate fraction, respectively. Also shown are
the translation products of the mRNA encoding residues 143-351 of
NIPP-1 in total lysates (lane 3) and in the heat-stable lysate
fraction (lane 4). Lane 5 shows a control translation
with no exogenous mRNA added.
Two further lines of evidence showed that the 29-kDa translation product resulted from initiation at the second ATG (bp 436-438), rather than from proteolysis of the 47-kDa species of NIPP-1. First, translation of an in vitro transcribed NIPP-1 mRNA that lacked 431 nucleotides at the 5`-end still resulted in the accumulation of the 29-kDa polypeptide (Fig. 4). Second, mutation of the first ATG codon in the full-length cDNA into an ATC completely abolished the synthesis of the 47-kDa product, but still yielded the 29-kDa polypeptide (not shown).
Figure 5:
Bacterial expression of an active fragment
of NIPP-1. Bacteria were transformed with the pBl-657 plasmid, which
contains an insert encoding residues 143-351 of NIPP-1. A shows a Coomassie staining of the purified recombinant polypeptide
after 12% Tricine-SDS-PAGE (lane 1). Lanes 2 and 3 show an autoradiogram of the purified recombinant fragment
after incubation with -
P-labeled MgATP in the absence (lane 2) or presence (lane 3) of protein kinase A
plus casein kinase 2. B shows the effect of the indicated
concentrations of the recombinant NIPP-1 fragment on the phosphorylase
phosphatase activity of PP-1
. The fragment was added as
such (
), after trypsinolysis (
), or after phosphorylation
by protein kinase A plus casein kinase 2 (
). The results
represent the means ± S.E. for three
experiments.
Several lines of
evidence suggest that the recombinant 15-kDa fragment of NIPP-1, which
probably roughly corresponds to the central third of NIPP-1, contains
the inhibitory domain. First, in agreement with our previous findings
for purified NIPP-1, the recombinant fragment inhibited the
phosphorylase phosphatase activity of PP-1, but was
destroyed by trypsin (Fig. 5B). Second, the fragment
was phosphorylated by protein kinase A and casein kinase 2 (Fig. 5A), and its inhibitory potency was decreased
severalfold by such phosphorylation (Fig. 5B). When
tested separately, protein kinase A turned out to be more efficient
than casein kinase 2 in inactivating the recombinant NIPP-1 fragment
(not illustrated).
Figure 6: Identification of NIPP-1 in subcellular liver fractions by Western analysis. A cytosolic fraction and a nuclear salt extract were prepared from rat liver as indicated in the ``Experimental Procedures.'' About 15 µg of protein of each fraction was subjected to 10% Tricine-SDS-PAGE, transferred to a membrane, and probed with antibodies against the COOH terminus of NIPP-1.
Remarkably, the migration of NIPP-1 also depended on the adopted electrophoresis system. Thus, we have noted that NIPP-1 rather migrates as a polypeptide of 41 kDa when glycine instead of Tricine is used as the trailing ion during SDS-PAGE(7) .
Figure 7:
Northern blot analysis in calf thymus and
in various human tissues. Each lane contains 2 µg of
poly(A) RNA isolated from the indicated tissue. The
blots were hybridized with a probe corresponding to bp 432-1088 of the
full-length NIPP-1 cDNA clone.
Western analysis only revealed a single
nuclear species of NIPP-1 with an apparent mass of 41 kDa (7) or 47 kDa (Fig. 6), depending on the adopted
electrophoresis system. We have previously shown that this polypeptide
is present as a complex with about half of all PP-1 that is
present in a nuclear extract(7) . Antibodies directed against
the COOH terminus of NIPP-1 did not detect fragments corresponding to
ard-1 or to alternative translational initiation products. This could
mean that such fragments are either not expressed in vivo or
that they are much less abundant than the 41-47-kDa fragments.
Alternatively, the generation of initiation or splice variants may be
cell type- or cell cycle-dependent. It cannot be excluded either that
other NIPP-1 species were not detected due to their rapid proteolysis
or posttranslational modification at the COOH terminus.
RNase E, ard-1,
and NIPP-1 also show a limited homology to the 70-kDa U1 RNA-associated
protein ((22) ; this work), which is a component of the U1
small nuclear ribonucleoprotein complex and is involved in RNA splicing
in eukaryotes(23) . Finally, a role for NIPP-1 in pre-mRNA
splicing is also indicated by observations that NIPP-1 is physically
associated with PP-1(7) , which has recently been
shown to modulate both spliceosome assembly as well as the splicing
process itself(24) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z50748[GenBank].