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J. Biol. Chem., Vol. 277, Issue 35, 31834-31841, August 30, 2002
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From the Afdeling Biochemie, Faculteit Geneeskunde, Katholieke
Universiteit Leuven, B-3000 Leuven, Belgium
Received for publication, May 7, 2002, and in revised form, June 12, 2002
NIPP1 is a ubiquitously expressed nuclear protein
that functions both as a regulator of protein Ser/Thr phosphatase-1 and as a splicing factor. The N-terminal part of NIPP1 consists of a
phosphothreonine-interacting Forkhead-associated (FHA) domain. We show
here that the FHA domain of NIPP1 interacts in vitro and in vivo with a TP dipeptide-rich fragment of the splicing
factor SAP155/SF3b155, a component of the U2 small nuclear
ribonucleoprotein particle. The NIPP1-SAP155 interaction was
entirely dependent on the phosphorylation of specific TP motifs in
SAP155. Mutagenesis and competition studies revealed that various
phosphorylated TP motifs competed for binding to the same site in the
FHA domain. The SAP155 kinases in cell lysates were blocked by
the Ca2+ chelator EGTA and by the
cyclin-dependent protein kinase inhibitor roscovitine. The
phosphorylation level of SAP155 was dramatically increased during
mitosis, and accordingly the activity of SAP155 kinases was augmented
in mitotic lysates. We discuss how the interaction between NIPP1 and
SAP155 could contribute to spliceosome (dis)assembly and the catalytic
steps of splicing.
Spliceosomes catalyze the removal of intronic sequences from
primary transcripts (pre-mRNAs) in two consecutive
transesterification reactions (1-3). Their major components are the
U1, U2, U5, and U4/U6 small nuclear ribonucleoprotein
particles
(snRNPs),1 which consist of
small nuclear RNAs and a set of snRNP proteins. These snRNPs are
recruited from nuclear storage/assembly sites known as the splicing
factor compartments or speckles, and their assembly on pre-mRNAs
occurs in a stepwise manner. According to a recent model of spliceosome
assembly (2), U1 snRNP and U4/U6·U5 tri-snRNP first contact the 5'
splice site. Subsequently U2 snRNP binds stably to the intronic branch
site region. The creation of splicing-competent spliceosomes also
depends on the recruitment of numerous non-snRNP-associated splicing
factors and requires multiple, ordered rearrangements between
spliceosomal components.
Substantial evidence implicates reversible protein phosphorylation in
both spliceosome (dis)assembly and splicing catalysis (2, 4). For
example, the SR family of splicing factors contain a domain that is
rich in Arg/Ser dipeptides, and the phosphorylation of these motifs by
specific "SR" kinases has been shown to modulate the interaction of
SR proteins with other splicing factors and with RNA during spliceosome
assembly (5). The shuttling of SR proteins between the speckles and the
spliceosomes and the dispersion of SR proteins from the speckles during
mitosis is also controlled by phosphorylation (6-8). The splicing
factor SF1/BBP, which interacts with U2AF65 in an early step of
spliceosome assembly, is a substrate for phosphorylation by protein
kinase G (9). The phosphorylation of SF1/BBP inhibits its interaction with U2AF65 and blocks splicing complex formation. A role for protein
phosphatases in pre-mRNA splicing has also been firmly established.
The protein Ser/Thr phosphatase PP2C The only splicing factor that is known to be phosphorylated during
splicing catalysis is SAP155, also named SF3b155 (14). This
protein is one of the five subunits of the SF3b complex that, together
with the SF3a protein complex, binds to a 12 S precursor of U2
snRNP and thereby converts it to the 17 S form that is
recruited to the spliceosomes (15, 16). SAP155 contacts pre-mRNA on
both sites of the branch site early in spliceosome assembly and
is thus positioned near the spliceosome catalytic center. SAP155 also
binds to U2AF35 (17), U2AF65 (17), cyclin E (18), and the SF3b
components SAP130 and p14 (15-17), although it appears unlikely that
these proteins all interact simultaneously with SAP155. It has been
suggested that the phosphorylation of SAP155 during splicing catalysis
somehow affects its association with RNA or with associated splicing
factors (14).
Our interest in the control of pre-mRNA splicing by protein
(de)phosphorylation stems from work on the nuclear protein NIPP1 (38.5 kDa). Originally we identified NIPP1 as an inhibitory subunit of a
nuclear species of the Ser/Thr-specific protein phosphatase PP1, hence
its name nuclear inhibitor of PP1
(19, 20). The heterodimeric complex between NIPP1 and PP1 is inactive,
but a protein phosphatase activity is revealed either by the binding of
NIPP1 to RNA (21) or by the phosphorylation of NIPP1 on specific Ser/Thr or Tyr residues (21, 22). NIPP1 is enriched in the nuclear
speckles (23, 24) but is also a component of the spliceosomes (25). In
nuclear extracts NIPP1 is required for spliceosome assembly, and
pre-mRNA splicing by these extracts is blocked when the recruitment
of NIPP1 to the spliceosomes is prevented. Remarkably, the function of
NIPP1 in spliceosome assembly is unrelated to its ability to bind PP1
or RNA, indicating that NIPP1 can act independently as a splicing
factor and as a protein phosphatase regulator.
The N-terminal third of NIPP1 largely consists of a Forkhead-associated
(FHA) domain (26), a phosphothreonine-binding module that is also
present in many other, mostly nuclear proteins (27, 28). The FHA domain
is required for the targeting of NIPP1 to the speckles (24) and to the
spliceosomes (25). We have previously shown that the FHA domain of
NIPP1 interacts with CDC5L, a regulator of pre-mRNA splicing and
mitotic entry (26). Although CDC5L is also present in the speckles, the
targeting of NIPP1 to the speckles could at most be partially accounted
for by its association with CDC5L since some speckles contained NIPP1
but no CDC5L. These data suggested that the FHA domain of NIPP1 has
additional binding partners. Accordingly we show here that the FHA
domain of NIPP1 also binds to the splicing factor SAP155 in
vitro and in vivo. We furthermore demonstrate that the
interaction between both components is controlled by multisite
phosphorylation and is cell cycle-regulated.
Materials--
Synthetic fragments of Xenopus
laevis SAP155
(109KIANREDEYKQQRRKMI125) and human NIPP1
(341PGKKPTPSLLI351) coupled to keyhole limpet
hemocyanin were used for the generation of rabbit polyclonal
antibodies. The antibodies were affinity-purified on the bovine serum
albumin-coupled peptides linked to CNBr-activated Sepharose 4B.
Monoclonal LexA tag and polyclonal His tag antibodies were purchased
from Santa Cruz Biotechnology. Rabbit and mouse secondary antibodies
were obtained from Dako. Human cyclin E-Cdk2 was produced by
baculovirus expression in High FiveTM cells (26). Cyclin
B-Cdk1, purified from X. laevis oocytes, was a
kind gift from Drs. J. Goris and I. Stevens. The catalytic subunit of
PP1 was prepared from rabbit skeletal muscle (26). Other key materials
were obtained from MBI Fermentas GmbH (restriction enzymes and
Pwo DNA polymerase), Sigma (Colcemid), Alexis
Biochemicals (roscovitine), Calbiochem (microcystin-LR), Roche
Diagnostics (endoproteinase Lys-C and Asp-N), and the Computer Cell
Culture Center (HeLa cell nuclear extracts).
Expression Vectors and Yeast Dual-hybrid
Screening--
Constructs for the bacterial expression of
polyhistidine-tagged SAP155-(1-491) and SAP155-(223-322) were
generated by subcloning of the corresponding PCR fragments of SAP155 in
the pET16b plasmid. Vectors for the bacterial expression of GST- or
polyhistidine-tagged NIPP1-(1-142) or NIPP1-(1-142) mutant
(S68A/R69A/V70A/H71A) were described before (24, 26). Point mutations
were introduced by the QuikChangeTM site-directed
mutagenesis method of Stratagene. For the yeast two-hybrid screening,
NIPP1-(1-142) and NIPP1-(225-351) were cloned in-frame with the LexA
protein (which includes a DNA-binding protein) in the pEG202 plasmid
(26). The NIPP1-(1-142) construct was used as bait for the screening
of a HeLa cell cDNA library subcloned in the pJG4-5 plasmid
in-frame with the B42 activation domain. For the mapping of the
interaction domain between SAP155 and NIPP1-(1-142) the SAP155
fragments 1-222, 323-491, 277-376, 201-301, 257-322, 257-301, and
381-491 were subcloned in-frame with the B42 activation domain in the
pJG4-5 plasmid. The Co-immunoprecipitations and GST Pull-downs--
Liver nuclear
extracts, used for pull-downs with GST-NIPP1-(1-142), were prepared as
described by Jagiello et al. (20) except that the extracts
were finally diluted in buffer without salt and Triton X-100. The
extracts were prepared in the absence or presence of protein
phosphatase inhibitors (0.5 µM microcystin-LR, 20 mM NaF, and 1 mM sodium vanadate). An aliquot
of the extracts prepared without phosphatase inhibitors was
preincubated for 10 min at 30 °C with PP1 (60 nM). For
some pull-down assays recombinant SAP155-(223-322) (5 µg) was first
phosphorylated by an overnight incubation at 30 °C in 30 µl of a
buffer containing 20 mM Tris-HCl at pH 7.4, 1 mM dithiothreitol, 4 mM magnesium acetate, 1 mM ATP, and 0.12 µg cyclin E-Cdk2. Immunoprecipitations,
GST pull-downs, and Western analyses were performed as described
previously (20, 26).
Splicing Assays--
A capped Determination of Cyclin E-Cdk2 Phosphorylation
Sites--
Radioactively phosphorylated SAP155-(223-322) (25 µg)
was precipitated with 20% (w/v) trichloroacetic acid. The pellet was washed once with 20% trichloroacetic acid and twice with diethyl ether
and then dissolved in 100 µl of a buffer containing 20 mM Tris at pH 8.5 and 0.01% (w/v) sodium dodecylsulfate. After an overnight incubation with 0.5 µg of endoproteinase Lys-C or
endoproteinase Asp-N at 37 °C in a total volume of 200 µl, the
digest was applied to a reversed-phase column (µRPC C2/C18 SC2.1/10
from Amersham Biosciences) equilibrated in 0.1% (v/v) trifluoroacetic
acid. The retained peptides were eluted with a linear gradient of
acetonitrile (0-70%) in 0.1% trifluoroacetic acid. The radioactive
fractions of the endoproteinase Asp-N digest were reapplied to the same reversed-phase column, this time equilibrated in water. The retained peptide was eluted with a linear gradient of ammonium acetate (0-10
mM) at pH 6.5. The radioactive peptides were sequenced as detailed before (21). The phosphopeptides obtained from the endoproteinase Lys-C digest were
241GSETpPGATPGS251 and
241GSETPGATpPGS251, where Tp represents
phosphothreonine. The endoproteinase Asp-N digest yielded the
peptide 302DTPGHGSGWAETpPRT316.
Cell Culture and Cell Fractionation--
COS-1 or HeLa cells
were grown on sterile 10-cm plates in Dulbecco's modified Eagle's
medium containing 10% (v/v) fetal calf serum (Invitrogen). The cells
of one plate were lysed by sonication in 0.25 ml of a buffer containing
20 mM Tris at pH 7.4, 0.3 M NaCl, 0.1% Triton
X-100, 1 mM dithiothreitol, 5 µg/ml leupeptin, 0.5 mM phenylmethanesulfonyl fluoride, 0.5 mM
benzamidine, 1 µM microcystin-LR, and 1 mM
orthovanadate. Cells were blocked in mitosis by the addition of
Colcemid (0.14 µg/ml) 20-24 h before lysis. Transcription was
arrested by the addition of actinomycin D (1 µg/ml) for 1 or 3 h.
Phosphorylation-dependent Interaction between NIPP1 and
SAP155--
The FHA domain of NIPP1 (residues 1-142) was used as bait
in a yeast two-hybrid screening of a HeLa cell library. Of 41 positive clones, 15 clones encoded three different fragments of the splicing factor SAP155 (Fig. 1B). These
SAP155 fragments interacted with the FHA domain with an affinity
similar to CDC5L-(258-614) as determined by liquid
The SAP155 fragments that interacted with NIPP1 all comprised (a part
of) the TP dipeptide-rich domain in the N-terminal third of SAP155
(Fig. 1, A and B). The largest SAP155 fragment
(residues 1-491) was also the best interactor of the FHA domain.
However, deletion of the N-terminal 208 residues, which lie outside the TP-rich domain, only marginally reduced the interaction with NIPP1. Fragments of the TP-rich domain interacted less well with
NIPP1-(1-142) than did the intact TP-rich domain, and generally the
strength of the interaction decreased with the size of the SAP155
fragments. Interestingly, the non-overlapping fragments
SAP155-(223-322) and SAP155-(323-491) both interacted with
NIPP1-(1-142), indicating that the TP-rich domain of SAP155 contains
multiple, independent interaction sites for NIPP1.
To further explore the relevance of the interaction between NIPP1 and
SAP155, we performed co-precipitation experiments. Following a
preincubation of nuclear extracts from rat liver (Fig.
2A) or HeLa cells (Fig.
2B) with a bacterially expressed GST fusion of NIPP1-(1-142), a GST pull-down of the fusion protein caused a co-precipitation of SAP155. Likewise, the immunoprecipitation of NIPP1
from HeLa cell nuclear extracts was associated with a co-immunoprecipitation of SAP155 (Fig. 2B). Various
observations indicated that the SAP155-NIPP1 interaction was critically
dependent on the phosphorylation of SAP155. Thus, when the FHA domain
was mutated (S68A/R69A/V70A/H71A) in residues that are part of a
conserved phosphate-binding loop (29), no co-precipitation of SAP155
was observed (Fig. 2, A and B). This mutated FHA
domain also failed to interact with SAP155 in a yeast dual-hybrid assay
(not shown). Pull-down experiments furthermore revealed that the
co-precipitation of GST-NIPP1-(1-142) and SAP155 was increased when
the liver nuclear extracts were prepared in the presence of a mixture
of protein phosphatase inhibitors, while their co-precipitation was
nearly completely abolished after a preincubation of the extracts with PP1 (Fig. 2A). On the other hand, an interaction between
NIPP1 and SAP155 in HeLa cell nuclear extracts was only detected after a preincubation of the extracts with MgATP, suggesting that the endogenous SAP155 had been completely dephosphorylated during the
preparation and/or storage of the extracts (Fig. 2B). In
this respect, it is worthy of note that the nuclear extracts from HeLa cells were at least 10-fold more concentrated than those from rat
liver, which favors a faster and more complete dephosphorylation by
endogenous protein phosphatases.
Mapping of the Phosphorylation Sites That Confer Binding of
SAP155-(223-322) to NIPP1--
The TP-rich domain of SAP155 (Fig.
1C) has been shown to be a substrate for phosphorylation by
cyclin E-Cdk2 (18). We indeed found that SAP155-(223-322) and
SAP155-(1-491) are excellent substrates for in vitro
phosphorylation by cyclin E-Cdk2 as well as cyclin B-Cdk1 (Fig.
3A and not shown). Moreover, a
co-precipitation of GST-NIPP1-(1-142) and SAP155-(223-322) was
dependent on the prior phosphorylation of the SAP155 fragment by cyclin
E-Cdk2 or cyclin B-Cdk1 (Fig. 3B). Again a co-precipitation
of both components was not seen when the FHA domain was mutated in its
phosphate-binding loop.
The above results indicated that the binding of SAP155 to NIPP1 might
be dependent on the phosphorylation of TP motifs. We have subsequently
studied in some detail the role of the TP dipeptide motifs in the
interaction between NIPP1-(1-142) and SAP155-(223-322), which only
contains 13 of the 29 TP motifs that are present in the TP-rich domain
of SAP155. The involvement of each of the 13 TP motifs of
SAP155-(223-322) in the interaction with NIPP1-(1-142) was studied by
site-directed mutagenesis. Unexpectedly, not one of the 13 threonine to alanine point mutations completely abolished the
interaction of SAP155-(223-322) with NIPP1-(1-142) as measured in a
dual-hybrid assay (Fig. 4A),
although some of the point mutants showed a significantly decreased
interaction. These data suggested that the SAP155-(223-322)-NIPP1
interaction was controlled by multisite phosphorylation.
To map the set of phosphorylation sites in SAP155-(223-322) that
determine its interaction with NIPP1, we have identified phosphorylation sites of cyclin E-Cdk2 by the sequencing of
proteolytically derived phosphopeptides (see "Experimental
Procedures"). Three phosphorylation sites were identified as
Thr244, Thr248, and Thr313. All
three sites are followed by a proline, although none of the sites
contains a basic residue at position +3 (Fig. 1C) as is
commonly seen for Cdk phosphorylation sites. The mutation of all three
sites into an alanine decreased the in vitro phosphorylation of SAP155-(223-322) by cyclin E-Cdk2 by only 79% (Fig.
5A) but completely abolished
the ability of SAP155-(223-322) to bind to GST-NIPP1-(1-142) in
vitro (Fig. 5B). Also, the triple mutant did not
interact with the FHA domain of NIPP1 in a dual-hybrid assay (Fig.
4A), although this mutant was expressed well (Fig. 4B). The mutation of two of the three mapped phosphorylation
sites only partially abolished the interaction of SAP155-(223-322)
with NIPP1-(1-142) (Fig. 4A). Importantly, the mutation of
Thr244, Thr248, and Thr313 did not
abolish the binding of a larger SAP155 fragment (SAP155-(1-491)) to
NIPP1 as determined by pull-downs with GST-NIPP1-(1-142) or by
two-hybrid assays (not shown). The latter data are in accordance with
our findings that the fragment of the TP-rich domain that is C-terminal
to residues 223-322 also harbors one or more binding sites for NIPP1
(Fig. 1B).
SAP155-derived Phosphopeptides Disrupt Complexes with
NIPP1--
The data in Fig. 4 suggested that SAP155-(223-322)
acquires a binding site for NIPP1 after phosphorylation of
Thr244, Thr248, or Thr313. To
distinguish between common or distinct NIPP1 binding sites for these
phosphothreonines, we have performed competition studies with synthetic
SAP155-derived phosphopeptides. Fig. 5C shows that the
synthetic peptide SAP155-(308-318), with Thr313
phosphorylated, inhibited the binding of SAP155-(223-322) to GST-NIPP1-(1-142) in a concentration-dependent way. No
competition was seen with the non-phosphorylated peptide. Combined with
our observations that the FHA domain that was mutated in the
established phosphate-binding loop failed to bind SAP155 (Figs. 2 and
3), these data strongly indicate that the FHA domain only contains a
single phosphothreonine binding site. Another phosphopeptide, SAP155-(239-253), with Thr244 and Thr248
phosphorylated, was a less efficient competitor (not shown), suggesting
that these phosphothreonines bind to NIPP1 with a lower affinity than
does phospho-Thr313.
The phosphopeptide SAP155-(308-318) (Thr(p)313)
also disrupted the interaction between NIPP1 and full-length SAP155 in
nuclear extracts in a concentration- and
phosphorylation-dependent manner (Fig. 5C).
These data suggested that all NIPP1 binding sites of SAP155, including
those outside residues 223-322 (Fig. 1B), interact with the
same fragment of NIPP1. Importantly, SAP155-(308-318) (Thr(p)313) also dissociated a complex of
GST-NIPP1-(1-142) and phosphorylated CDC5L (not shown), indicating
that SAP155 and CDC5L compete for the same binding site in the FHA
domain of NIPP1.
Since the phosphopeptide SAP155-(308-318) (Thr(p)313)
disrupted the interaction between the FHA domain of NIPP1 and its
ligands in nuclear extracts (Fig. 5C), we have explored
whether this peptide also interfered with the recruitment of NIPP1 to
the spliceosomes and with pre-mRNA splicing. Surprisingly, while
the addition of the FHA domain of NIPP1 blocked the
co-immunoprecipitation of endogenous NIPP1 and radioactively labeled
SAP155 Is Hyperphosphorylated during Mitosis--
Since SAP155
acquired binding sites for NIPP1 after phosphorylation by an interphase
Cdk (cyclin E-Cdk2) as well as by a mitotic Cdk (cyclin B-Cdk1), we
have examined whether the phosphorylation of SAP155 is regulated in a
cell-cycle dependent manner. Pull-down assays with GST-NIPP1-(1-142)
showed a huge increase in the amount of SAP155 that co-sedimented from
lysates prepared from mitotically arrested COS-1 cells (Fig.
7A) or HeLa cells (not shown)
as compared with that from asynchronously dividing (interphase) cells.
The increased binding of SAP155 in mitotic extracts was not due to an
increased total concentration of SAP155 (Fig. 7B) and
required an FHA domain with an intact phosphate-binding loop (Fig.
7A), suggesting that it was mediated by an increased
phosphorylation of SAP155. On the other hand, the amount of SAP155 that
co-immunoprecipitated with NIPP1 was the same in interphase and mitotic
lysates (Fig. 7C). Collectively these data suggested that
SAP155 and NIPP1 are part of the same macromolecular complex during
interphase as well as during mitosis, but during mitosis, SAP155 is
hyperphosphorylated on sites that mediate binding to NIPP1. It remains
to be examined whether the direct interaction between SAP155 and NIPP1
is actually increased during mitosis or whether SAP155 is merely primed
for binding to NIPP1 after mitosis.
Characterization of SAP155 Kinases--
To gain additional insight
into the nature of the protein kinase(s) that phosphorylate SAP155, we
have examined the effect of protein kinase inhibitors on the
phosphorylation of SAP155-(1-491) by cell lysates from asynchronously
dividing and mitotically arrested cells. We noted that the
SAP155-(1-491) kinase activity in lysates from mitotically arrested
cells (Fig. 8B) was about
2-fold higher than that in lysates from asynchronously dividing cells
(Fig. 8A) as expected from the increased binding of SAP155
to GST-NIPP1 in mitotic lysates (Fig. 7A). Inhibitors of
protein kinase C (bisindolylmaleimide I), protein kinase A (protein
kinase A inhibitory peptide), mitogen-activated protein kinases
(PD98059 and PD1693168), glycogen synthase kinase 3 (LiCl),
phosphatidylinositol 3-kinase (wortmannin), and protein kinase CK2
(heparin) did not measurably affect the SAP155 kinase activity (not
shown). However, the Ca2+ chelator EGTA blocked the SAP155
kinase activity in lysates from asynchronously dividing and mitotically
arrested cells by about 90 and 70%, respectively (Fig. 8). On the
other hand, roscovitine, an established Cdk inhibitor (30), decreased
the SAP155 kinase activity in mitotic and interphase lysates by about
50 and 30%, respectively. The higher contribution of a
roscovitine-sensitive kinase in mitotic lysates is in accordance with a
role for Cdk1 as a major mitotic SAP155 kinase (see "Discussion").
The addition of both EGTA and roscovitine blocked nearly all the SAP155
kinase activity in cell lysates (Fig. 8). Importantly, EGTA and
roscovitine also decreased the binding of SAP155-(1-491) to NIPP1 in
pull-down assays (Fig. 8B, inset), indicating
that the inhibited kinase(s) catalyzes the phosphorylation of sites in
SAP155 that control its affinity for NIPP1.
The N-terminal domain of SAP155 contains two putative
phosphorylation sites (Thr125 and Ser217) for
the Ca2+- and calmodulin-dependent protein
kinase II. Since EGTA efficiently blocked the phosphorylation of SAP155
by cell lysates (Fig. 8), we have explored whether this protein kinase
could contribute to the SAP155-NIPP1 interaction. We found that
recombinant Ca2+-calmodulin-dependent protein
kinase II was indeed an efficient SAP155 kinase in vitro but
did not endow SAP155-(1-491) with a high affinity binding site for
NIPP1 (not shown). Thus, the Ca2+-dependent
phosphorylation of SAP155 in cell lysates is either mediated by another
Ca2+-dependent protein kinase or by a protein
kinase that is activated by a Ca2+-dependent process.
Binding Determinants of the FHA Domain of NIPP1--
FHA domains
have been identified as phosphoprotein binding modules, but the exact
determinants of their interaction are only partially known (27, 29).
They bind preferentially to phosphothreonine-containing peptides,
although one of the FHA domains of Rad53 has also been shown to bind to
peptides with a phosphotyrosine (31). Studies using peptide library
screening revealed that motif selection typically extends for three
residues N-terminal and C-terminal to the phosphothreonine. Of
particular importance for the binding specificity of various FHA
domains is the residue in the Thr(p) +3 position (27, 29). However,
this may not be true for the FHA domain of NIPP1 since the three mapped
phosphothreonines that mediate binding of SAP155 to NIPP1 are followed
by a different residue (Ala, Ser, or Thr) at position +3 (Fig.
1C). On the other hand, these phosphothreonines are all
three followed by a proline, which therefore may represent an important
determinant of binding selectivity. This view is in accordance with
previous findings that the interaction of NIPP1 with CDC5L is also
mediated by a TP-rich domain (26). The domain of SAP155 that interacts
with NIPP1 (this work) contains, in addition to multiple TP motifs, also various RWD and GH motifs (14). However, it seems unlikely that
these motifs are important determinants for the interaction with NIPP1
since such motifs are not present in CDC5L.
The binding specificity of the FHA domain of NIPP1 is most similar to
that of the Ki-67 antigen. The latter FHA domain binds to the nucleolar
RNA-binding protein NIFK, and this interaction involves two
phosphorylated TP motifs (32). However, the mutation of only one of
these TP motifs abolished the binding of NIFK to Ki-67 in a
two-hybrid assay. This is different for the SAP155-(223-322)-NIPP1 interaction, which was only abolished after mutation of at least three
Cdk2-phosphorylated TP motifs (Fig. 4), pointing to the presence of
multiple, independent binding sites for NIPP1. It seems likely that
these TP motifs all interact with NIPP1 in a similar or identical
manner since the SAP155-NIPP1 complex could be completely disrupted
with a synthetic peptide that contained only one of these
(phosphorylated) TP motifs (Fig. 5C). That the FHA domain of
NIPP1 only has a single phosphothreonine binding site is also in
accordance with findings that mutation of residues 68-71, which are
part of a phosphate-binding loop that is conserved in all FHA domains,
completely abolished the interaction with SAP155 (Fig. 3).
An intriguing question concerns the need for multiple independent and
mutually exclusive NIPP1 binding sites in SAP155. It is possible that
multiple binding sites increase the stability of the NIPP1-SAP155
complex by decreasing the rate of dissociation. It can also be
envisaged that the NIPP1 binding sites are not equally accessible
in vivo, e.g. because their phosphorylation is
differently controlled or because binding of NIPP1 is hampered by
competing ligands.
Both SAP155 (3, 15) and CDC5L (33) are subunits of large protein
complexes. Our finding that the binding of NIPP1 to these proteins is
phosphorylation-dependent explains why NIPP1 has previously
not been recognized as a subunit of these complexes since no
precautions were taken to prevent dephosphorylation reactions during
the purification procedure.
SAP155 Kinases--
Various lines of evidence point to a role of
cell cycle-regulated protein kinases in the control of the SAP155-NIPP1
interaction in mammalian cells. Thus, cyclin B-Cdk1 and Cyclin E-Cdk2
acted as SAP155 kinases in vitro (Fig. 3), and roscovitine,
an established Cdk inhibitor, inhibited the SAP155 kinase activity in
cell lysates (Fig. 8). Also, cyclin E-Cdk2 has been shown to directly
associate with the TP-rich domain of SAP155 (18), and the SAP155 kinase activity in cell lysates was doubled during mitosis when Cdk1 is
activated (Fig. 8). Interestingly, the splicing factor CDC5L, another
interactor of the FHA domain of NIPP1 (26), has also been identified as
a mitotic phosphoprotein (34).
It is intriguing that roscovitine also inhibited the SAP155 kinase
activity in interphase extracts when Cdk2 is only transiently active.
This could indicate that other interphase Cdks are also implicated in
the phosphorylation of SAP155. An obvious candidate would be Cdk7,
which is also nuclear and is sensitive to inhibition by roscovitine
(30). On the other hand, the SAP155 kinase activity was only partially
inhibited by roscovitine (Fig. 8), indicating that the TP domain of
SAP155 can also be phosphorylated by a cyclin-independent protein
kinase(s). This conclusion is in accordance with the report that a
large fraction of the protein kinase activity in SAP155 immunoprecipitates is insensitive to a Cdk inhibitor (18). We found
that the SAP155 kinase activity in cell lysates is efficiently inhibited by EGTA (Fig. 8), suggesting that SAP155 may be a substrate for a Ca2+-dependent protein kinase. Two
members of the family of Ca2+- and
calmodulin-dependent protein kinases are nuclear and
therefore qualify as potential SAP155 kinases, i.e.
Ca2+- and calmodulin-dependent protein kinase IV
(35) and PSKH1 (36). Interestingly, PSKH1 shows a dotted
nuclear distribution, which could point to an association with the
speckles. Further experiments are needed to explore the contribution of
a Ca2+-dependent protein kinase(s) in the
phosphorylation of SAP155. It should also be pointed out that
Ca2+-dependent kinases may interfere indirectly
with the phosphorylation of SAP155, for example by affecting the
activity of Cdks. Thus, it has been reported that Ca2+- and
calmodulin-dependent protein kinase II phosphorylates and activates
Cdc25, which acts as a final trigger for activation of Cdk1 (37).
Finally, we cannot entirely rule out the possibility that
the phosphorylation of SAP155 in cell lysates is mediated by a novel
type of protein kinase that is inhibited by both EGTA and roscovitine
and is activated during mitosis. However, it would be difficult to
understand why such a kinase is inhibited more by roscovitine in
mitotic lysates than in interphase lysates (Fig. 8).
In retrospect it is somewhat surprising that we picked up SAP155 as a
phosphorylation-dependent interactor of NIPP1 in a yeast dual-hybrid screening since Saccharomyces cerevisiae lacks a
NIPP1 homolog and its SAP155 homolog (Hsh155p) lacks a TP-rich domain (38, 39). Nevertheless, the results of our dual-hybrid screening show
that yeast contains a protein kinase(s) that phosphorylates mammalian
SAP155 on a site(s) that confers binding to NIPP1.
Role of the NIPP1-SAP155 Interaction--
The targeting of NIPP1
to the nuclear speckles (24) and to the spliceosomes (25) is mediated
by its FHA domain. This explains why the recruitment of NIPP1 to the
spliceosomes in nuclear extracts can be competitively blocked by the
addition of an excess of the FHA domain (Fig. 6A).
Mutagenesis studies have revealed that the phosphate-binding loop in
the FHA domain is essential for the targeting of NIPP1 to the speckles
and to the spliceosomes (24, 25). Since this phosphate-binding loop is
also essential for the interaction of NIPP1 with the splicing factors
SAP155 and CDC5L, we speculated that NIPP1 is anchored to the speckles
and to the spliceosomes via phosphorylated forms of these splicing factors. Yet a SAP155-derived phosphopeptide that disrupted the interaction between NIPP1 and SAP155 or CDC5L in nuclear extracts did
not interfere with the recruitment of NIPP1 to the spliceosomes. These
data indicate that NIPP1 is recruited to the spliceosomes independently
from SAP155 and CDC5L, which is in agreement with our previous report
that SAP155 is recruited at an earlier stage of spliceosome assembly
than is NIPP1 (25). Our finding that the association of NIPP1 with the
spliceosomes is disrupted by the addition of the FHA domain but not by
one of its phosphorylated ligands also suggests that the FHA domain may
contain additional interaction sites for spliceosomal components
outside the phosphate-binding loop. An alternative explanation is that
the phosphate-binding loop of the FHA domain interacts in the
spliceosomes with yet another phosphorylated splicing factor. If the
latter could bind to the FHA domain with a much higher affinity than
did SAP155 or CDC5L, this could explain our inability to disrupt this
complex with the SAP155-derived phosphopeptide.
We can currently only speculate on the possible function of the
NIPP1-SAP155 interaction during spliceosome (dis)assembly and splicing
catalysis. When the recruitment of NIPP1 to the spliceosomes is
prevented, spliceosome assembly is blocked at the transition between
the B and C complex (25). This could indicate that NIPP1 is involved in
rearrangements between SAP155 and its interactors at the last stages of
spliceosome assembly. In this respect it is worthy to note that, in
addition to NIPP1 (this work), also cyclin E (18), U2AF65 (17), and p14
(16) have been reported to interact with the TP-rich domain of SAP155.
It would be interesting to explore whether the interactions with cyclin
E, U2AF65, and p14 are also phosphorylation-dependent and
whether these interactions are affected by the binding of NIPP1.
It is also possible that NIPP1 targets SAP155 or its associated
proteins for dephosphorylation by PP1. However, since PP1 does not
appear to play an essential role in spliceosome assembly and splicing
catalysis in nuclear extracts (25), we suggest that the
dephosphorylation of SAP155 or its associated proteins is rather
involved in spliceosome disassembly and/or the targeting of splicing
factors to the speckles. By a similar mechanism the increased affinity
of SAP155 for NIPP1 (Fig. 8) may contribute to the silencing of
splicing before mitosis. The proposed substrate-targeting function of
NIPP1 is similar to the role of many other non-catalytic subunits of
PP1 (40). Furthermore, it should be pointed out that the
dephosphorylation of splicing factors of the SR family by PP1 has also
been implicated in spliceosome disassembly and in their shuttling to
the speckles (7). Thus, the subnuclear targeting of SR proteins and
unrelated splicing factors, such as SAP155, may be regulated by
phosphorylation. It is currently unclear which sites of SAP155 may be
targeted for dephosphorylation by NIPP1-associated PP1. Obvious
candidates are the sites of SAP155 that are phosphorylated during
splicing (14).
In conclusion, we have shown here that NIPP1 interacts in
vitro and in vivo with the splicing factor SAP155 and
that this interaction is critically dependent on the cell
cycle-regulated phosphorylation of specific TP dipeptide sequences in
the N-terminal domain of SAP155. Further investigations are needed to
explore how the interaction between NIPP1 and SAP155 contributes to
spliceosome (dis)assembly and/or the catalytic steps of splicing.
Dr. V. Vulsteke is acknowledged for expert
advice on cell culture. We thank V. Feytons for the synthesis of
(phospho)peptides. Nicole Sente and Karolien Nelissen provided expert
technical assistance.
*
This work was supported by Fund for Scientific
Research-Flanders Grant G.0374.01 and by a Flemish Concerted Research
Action.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
§
A postdoctoral fellow of the National Fund for Scientific
Research-Flanders.
¶
To whom correspondence should be addressed: Afdeling
Biochemie, Campus Gasthuisberg, Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-34-57-01; Fax: 32-16-34-59-95; E-mail:
Mathieu.Bollen@med.kuleuven.ac.be.
Published, JBC Papers in Press, June 24, 2002, DOI 10.1074/jbc.M204427200
The abbreviations used are:
snRNP, small nuclear
ribonucleoprotein particle;
FHA, Forkhead-associated;
PP, protein
phosphatase;
NIPP1, nuclear inhibitor of PP1;
GST, glutathione
S-transferase;
NIFK, nucleolar protein interacting with the
FHA domain of Ki-67 antigen;
PSKH1, protein serine kinase with homology
to Ca2+- and calmodulin-dependent kinase
I.
Phosphorylation-dependent Interaction between the
Splicing Factors SAP155 and NIPP1*
,§,
ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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is required for an early step
of spliceosome assembly (10), while okadaic acid-sensitive protein
phosphatases, such as PP2A, appear to function during splicing
catalysis (11). The adenovirus E4-ORF4 protein induces the
dephosphorylation of host cell SR proteins by the recruitment of PP2A,
which alleviates the inhibition of adenovirus IIIa pre-mRNA
splicing by the SR proteins (12, 13).
EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-galactosidase activities were quantified by a
liquid culture assay (CLONTECH yeast protocols handbook).
-globin pre-mRNA fragment,
comprising exon 1 through the BamHI site in exon 2, was
synthetized in the presence of [
-32P]GTP. This primary
transcript was used as a substrate for splicing in HeLa cell nuclear
extracts (25). Immunoprecipitations during the splicing reactions were
done as described previously (25).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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-galactosidase
assays. However, the SAP155 fragments did not interact with the
C-terminal domain of NIPP1 or with Sds22, an unrelated regulator of
protein phosphatase-1 (Fig. 1B and not shown).
View larger version (39K):
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Fig. 1.
SAP155 interacts with NIPP1-(1-142) in a
yeast two-hybrid assay. A, schematic representation of
the domain structure of human SAP155. The numbers
beneath the bar refer to amino acids of human
SAP155. B, interaction between NIPP1-(1-142) and SAP155 in
a yeast two-hybrid assay. CDC5L-(258-614) was included as a positive
control. The "empty" prey vector, encoding only the transactivation
domain, and the bait NIPP1-(225-351) were used as negative controls.
The numbers represent the -galactosidase activity
(means ± S.E., n = 5) as derived from a liquid
culture assay. The asterisks refer to the SAP155 fragments
that were originally picked up during the two-hybrid screening. The
SAP155 fragments that showed a strong interaction with NIPP1-(1-142)
in the two-hybrid assay are indicated in black. Weakly
interacting SAP155 fragments are indicated in gray.
C, primary structure of the TP-rich domain of human SAP155.
The TP dipeptide motifs are indicated in bold, and
established in vitro phosphorylation sites for cyclin E-Cdk2
are underlined. The numbers on the right refer to
amino acids of human SAP155.
View larger version (26K):
[in a new window]
Fig. 2.
Phosphorylation-dependent
interaction of SAP155 and NIPP1 in nuclear extracts. A,
co-precipitation of SAP155 and (mutated) GST-NIPP1-(1-142) from rat
liver nuclear extracts. The extracts were prepared in the absence or
presence of a mixture of inhibitors of protein phosphatases. An aliquot
of the extracts without phosphatase inhibitors was also preincubated
with PP1. The SAP155 that co-sedimented with the GST fusion proteins
was visualized by Western blotting with SAP155 antibodies.
B, HeLa cell nuclear extracts were incubated at 30 °C for
the indicated times with MgCl2 (4 mM) and ATP
(0.5 mM) to allow phosphorylation of the endogenous SAP155.
During the subsequent immunoprecipitation with NIPP1 antibodies
(upper panel) or pull-down with (mutated) GST-NIPP1-(1-142)
(lower panel), further protein (de)phosphorylation was
stopped by the addition of 10 mM EDTA, 1 µM microcystin-LR, and 1 mM orthovanadate.
IP, immunoprecipitation.
View larger version (14K):
[in a new window]
Fig. 3.
NIPP1-(1-142) interacts with recombinant
SAP155-(223-322) that is phosphorylated by cyclin E-Cdk2.
A, autoradiogram of SAP155-(223-322) phosphorylated by
cyclin E-Cdk2 in the presence of -32P-labeled ATP. In
the control condition cyclin E-Cdk2 was not included. B,
pull-down of SAP155-(223-322) with GST, GST-NIPP1-(1-142), or
GST-NIPP1-(1-142) mutant. The SAP155 fragment was preincubated under
phosphorylation conditions in the absence or presence of cyclin E-Cdk2.
The sedimented SAP155-(223-322) was visualized by Western blotting
with His tag antibodies.
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Fig. 4.
Mapping of the TP motifs of SAP155-(223-322)
that are involved in the binding to NIPP1. A, the
interaction of SAP155-(223-322) and the indicated point mutants with
NIPP1-(1-142) was quantified by liquid yeast dual-hybrid assays. The
gray bars represent -galactosidase activities (means ± S.E., n = 13). The asterisks indicate
mutants that interacted significantly less than wild type
SAP155-(223-322) (unpaired Student's t test;
p < 0.001). B, the expression of
SAP155-(223-322) and its mutants in a representative experiment as
detected by Western blotting with hemagglutinin tag antibodies.
wt, wild type.
View larger version (25K):
[in a new window]
Fig. 5.
The interaction between purified
SAP155-(223-322) and GST-NIPP1-(1-142) is abolished by mutagenesis or
by the addition of phosphopeptides. A, autoradiogram of
SAP155-(223-322) and triple mutated SAP155-(223-322)
(T244A/T248A/T313A) after phosphorylation with cyclin E-Cdk2 in the
presence of [ -32P]ATP. B, binding of
GST-NIPP1-(1-142) to wild type (Wt) and triple mutated
SAP155-(223-322) incubated without or with cyclin E-Cdk2. The
co-sedimented SAP155-(223-322) was detected by Western blot analysis
with His tag antibodies. C, the left panel shows
a pull-down of cyclin E-Cdk2-phosphorylated SAP155-(223-322) with
GST-NIPP1-(1-142) following a preincubation (30 min at 4 °C) of
GST-NIPP1-(1-142) with the indicated concentrations of the synthetic
peptide SAP155-(308-318) with Thr313 either phosphorylated
(pT313) or non-phosphorylated. The right panel
shows pull-downs of SAP155 from a HeLa cell nuclear extract with
GST-NIPP1-(1-142). The extracts were preincubated for 20 min at
30 °C under conditions of phosphorylation as detailed in the legend
of Fig. 1B. The pull-downs were done after a preincubation
of GST-NIPP1-(1-142) with phosphorylated (pT313) or
unphosphorylated SAP155-(308-318) for 30 min at 4 °C. The final
concentrations of the peptides, after the addition of the nuclear
extract, are indicated. SAP155 was visualized by Western blot analysis.
pT313, Thr(p)313.
-globin pre-mRNA, the addition of the phosphopeptide did not
have this effect (Fig. 6A).
Likewise, the FHA domain blocked pre-mRNA splicing of the -globin pre-mRNA fragment, but the SAP155-derived phosphopeptide did not interfere with splicing (Fig. 6B). Thus, the
recruitment of NIPP1 to the spliceosomes is independent of the
recruitment of SAP155 and CDC5L.
View larger version (25K):
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Fig. 6.
Phosphorylated SAP155-(308-318) does not
affect the recruitment of NIPP1 to the spliceosomes and does not
interfere with pre-mRNA splicing. A shows the
co-immunoprecipitation of NIPP1 and 32P-labeled -globin
pre-mRNA from in vitro splicing reactions. The
immunoprecipitations were done in control conditions or after the
addition of 10 µM NIPP1-(1-142) (FHA domain) or 750 µM SAP155-(308-318) (pT313). The open
bars represent the corresponding control immunoprecipitations,
i.e. without addition of the primary antibodies.
B, splicing of radioactively labeled -globin pre-mRNA
was carried out at 30 °C in the absence (control) or
presence of 10 µM NIPP1-(1-142) (FHA
domain) or in the presence of 750 µM
SAP155-(308-318) (pT313). After 90 min, the spliced
products were separated on a 9% denaturing polyacrylamide gel and
visualized by autoradiography. The splicing products are identified
schematically. IP, immunoprecipitation; Ab,
antibody; pT313, Thr(p)313.
View larger version (16K):
[in a new window]
Fig. 7.
The phosphorylation of SAP155 is cell
cycle-regulated. A, lysates from asynchronically
dividing (interphase lysates) or mitotically arrested COS-1 cells were
prepared in the presence of protein phosphatase inhibitors. The lysates
were used as a source of SAP155 for pull-down experiments with wild
type or mutated GST-NIPP1-(1-142). The sedimented SAP155 was detected
by Western blotting. B, Western blot of total lysate with
SAP155 antibodies. The minor bands between 72.5 and 100.1 kDa did not
co-sediment with GST-NIPP1-(1-142) (see A) and may
therefore represent aspecific polypeptides that are recognized by the
SAP155 antibodies. C, the lysates were also used to quantify
co-immunoprecipitation of SAP155 and NIPP1 with NIPP1 antibodies.
SAP155 in the immunoprecipitates was quantified by Western
analysis. Ab, antibody.
View larger version (32K):
[in a new window]
Fig. 8.
Inhibition of SAP155 kinases in cell
lysates. Interphase (A) or mitotic cell lysates
(B) were used as a source of protein kinases to
phosphorylate SAP155-(1-491) (60 min at 30 °C) in the presence of 1 mM -32P-labeled ATP and 2 mM
MgCl2. Phosphorylation reactions were done in the absence
(control) or presence of 1 mM EGTA and/or
15 µM roscovitine. Following SDS-PAGE, SAP155-(1-491)
was cut out of the gel, and the associated radioactivity was counted.
The figure shows the phosphorylation levels of SAP155-(1-491) as a
percentage (means ± S.E., n = 3) of the level
detected in the control condition with interphase lysates. The
inset shows a pull-down of SAP155-(1-491) with
GST-NIPP1-(1-142) after phosphorylation by mitotic lysates in the
absence and presence of roscovitine or EGTA.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
Both authors contributed equally to this work.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Das, R.,
Zhou, Z.,
and Reed, R.
(2000)
Mol. Cell
5,
779-787[CrossRef][Medline]
[Order article via Infotrieve]
2.
Hastings, M. L.,
and Krainer, A. R.
(2001)
Curr. Opin. Cell Biol.
13,
302-309[CrossRef][Medline]
[Order article via Infotrieve]
3.
Will, C. L.,
and Lührmann, R.
(2001)
Curr. Opin. Cell Biol.
13,
290-301[CrossRef][Medline]
[Order article via Infotrieve]
4.
Murray, M. V.
(1999)
in
Post-transcriptional Processing and the Endocrine System
(Chew, S. L., ed), Vol. 25
, pp. 83-100, Karger, Basel
5.
Xiao, S.-H.,
and Manley, J. L.
(1998)
EMBO J.
17,
6359-6367[CrossRef][Medline]
[Order article via Infotrieve]
6.
Misteli, T.,
and Spector, D. L.
(1996)
Mol. Biol. Cell
7,
1559-1572[Abstract]
7.
Misteli, T.,
and Spector, D. L.
(1997)
Trends Cell Biol.
7,
135-138[CrossRef][Medline]
[Order article via Infotrieve]
8.
Misteli, T.,
Cáceres, J. F.,
Clement, J. Q.,
Krainer, A. R.,
Wilkinson, M. F.,
and Spector, D. L.
(1998)
J. Cell Biol.
143,
297-307 9.
Wang, X.,
Bruderer, S.,
Rafi, Z.,
Xue, J.,
Milburn, P. J.,
Krämer, A.,
and Robinson, P. J.
(1999)
EMBO J.
18,
4549-4559[CrossRef][Medline]
[Order article via Infotrieve]
10.
Murray, M. V.,
Kobayashi, R.,
and Krainer, A. R.
(1999)
Genes Dev.
13,
87-97 11.
Mermoud, J. E.,
Cohen, P.,
and Lamond, A. I.
(1992)
Nucleic Acids Res.
20,
5263-5269 12.
Estmer Nilsson, C.,
Petersen-Mahrt, S.,
Durot, C.,
Shtrichman, R.,
Krainer, A. R.,
Kleinberger, T.,
and Akusjärvi, G.
(2001)
EMBO J.
20,
864-871[CrossRef][Medline]
[Order article via Infotrieve]
13.
Kanopka, A.,
Mühlemann, O.,
Petersen-Mahrt, S.,
Estmer, C.,
Öhrmalm, C.,
and Akusjärvi, G.
(1998)
Nature
393,
185-187[CrossRef][Medline]
[Order article via Infotrieve]
14.
Wang, C.,
Chua, K.,
Seghezzi, W.,
Lees, E.,
Gozani, O.,
and Reed, R.
(1998)
Genes Dev.
12,
1409-1414 15.
Das, B. K.,
Xia, L.,
Palandjian, L.,
Gozani, O.,
Chyung, Y.,
and Reed, R.
(1999)
Mol. Cell. Biol.
19,
6796-6802 16.
Will, C. L.,
Schneider, C.,
MacMillan, A. M.,
Katopodis, N. F.,
Neubauer, G.,
Wilm, M.,
Lührmann, R.,
and Query, C. C.
(2001)
EMBO J.
20,
4536-4546[CrossRef][Medline]
[Order article via Infotrieve]
17.
Gozani, O.,
Potashkin, J.,
and Reed, R.
(1998)
Mol. Cell. Biol.
18,
4752-4760 18.
Seghezzi, W.,
Chua, K.,
Shanahan, F.,
Gozani, O.,
Reed, R.,
and Lees, E.
(1998)
Mol. Cell. Biol.
18,
4526-4536 19.
Beullens, M.,
Van Eynde, A.,
Stalmans, W.,
and Bollen, M.
(1992)
J. Biol. Chem.
267,
16538-16544 20.
Jagiello, I.,
Beullens, M.,
Stalmans, W.,
and Bollen, M.
(1995)
J. Biol. Chem.
270,
17257-17263 21.
Beullens, M.,
Vulsteke, V.,
Van Eynde, A.,
Jagiello, I.,
Stalmans, W.,
and Bollen, M.
(2000)
Biochem. J.
352,
651-658[CrossRef][Medline]
[Order article via Infotrieve]
22.
Vulsteke, V.,
Beullens, M.,
Waelkens, E.,
Stalmans, W.,
and Bollen, M.
(1997)
J. Biol. Chem.
272,
32972-32978 23.
Trinkle-Mulcahy, L.,
Ajuh, P.,
Prescott, A.,
Claverie-Martin, F.,
Cohen, S.,
Lamond, A. I.,
and Cohen, P.
(1999)
J. Cell Sci.
112,
157-168[Abstract]
24.
Jagiello, I.,
Van Eynde, A.,
Vulsteke, V.,
Beullens, M.,
Boudrez, A.,
Keppens, S.,
Stalmans, W.,
and Bollen, M.
(2000)
J. Cell Sci.
113,
3761-3768[Abstract]
25.
Beullens, M.,
and Bollen, M.
(2002)
J. Biol. Chem.
277,
19855-19860 26.
Boudrez, A.,
Beullens, M.,
Groenen, P.,
Van Eynde, A.,
Vulsteke, V.,
Jagiello, I.,
Murray, M.,
Krainer, A. R.,
Stalmans, W.,
and Bollen, M.
(2000)
J. Biol. Chem.
275,
25411-25417 27.
Li, J.,
Lee, G.,
Van Doren, S. R.,
and Walker, J. C.
(2000)
J. Cell Sci.
113,
4143-4149[Abstract]
28.
Yaffe, M. B.,
and Elia, A. E. H.
(2001)
Curr. Opin. Cell Biol.
13,
131-138[CrossRef][Medline]
[Order article via Infotrieve]
29.
Durocher, D.,
Taylor, I. A.,
Sarbassova, D.,
Haire, L. F.,
Westcott, S. L.,
Jackson, S. P.,
Smerdon, S. J.,
and Yaffe, M. B.
(2000)
Mol. Cell
6,
1169-1182[CrossRef][Medline]
[Order article via Infotrieve]
30.
Ljungman, M.,
and Paulsen, M. T.
(2001)
Mol. Pharmacol.
60,
785-789 31.
Wang, P.,
Byeon, I.-J. L.,
Liao, H.,
Beebe, K. D.,
Yongkiettrakul, S.,
Pei, D.,
and Tsai, M.-D.
(2000)
J. Mol. Biol.
302,
927-940[CrossRef][Medline]
[Order article via Infotrieve]
32.
Takagi, M.,
Sueishi, M.,
Saiwaki, T.,
Kametaka, A.,
and Yoneda, Y.
(2001)
J. Biol. Chem.
276,
25386-25391 33.
Ajuh, P.,
Kuster, B.,
Panov, K.,
Zomerdijk, J. C. B. M.,
Mann, M.,
and Lamond, A. I.
(2000)
EMBO J.
19,
6569-6581[CrossRef][Medline]
[Order article via Infotrieve]
34.
Stukenberg, P. T.,
Lustig, K. D.,
McGarry, T. J.,
King, R. W.,
Kuang, J.,
and Kirschner, M. W.
(1997)
Curr. Biol.
7,
338-348[CrossRef][Medline]
[Order article via Infotrieve]
35.
Soderling, T. R.
(1999)
Trends Biochem. Sci.
24,
232-236[CrossRef][Medline]
[Order article via Infotrieve]
36.
Brede, G.,
Solheim, J.,
Tröen, G.,
and Prydz, H.
(2000)
Genomics
70,
82-92[CrossRef][Medline]
[Order article via Infotrieve]
37.
Patel, R.,
Holt, M.,
Philipova, R.,
Moss, S.,
Schulman, H.,
Hidaka, H.,
and Whitaker, M.
(1999)
J. Biol. Chem.
274,
7958-7968 38.
Habara, Y.,
Urushiyama, S.,
Tani, T.,
and Ohshima, Y.
(1998)
Nucleic Acids Res.
26,
5662-5669 39.
Haynes Pauling, M.,
McPheeters, D.,
and Ares, M., Jr.
(2000)
Mol. Cell. Biol.
20,
2176-2185 40.
Bollen, M.
(2001)
Trends Biochem. Sci.
26,
426-431[CrossRef][Medline]
[Order article via Infotrieve]
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 A. Van Eynde, M. Nuytten, M. Dewerchin, L. Schoonjans, S. Keppens, M. Beullens, L. Moons, P. Carmeliet, W. Stalmans, and M. Bollen The Nuclear Scaffold Protein NIPP1 Is Essential for Early Embryonic Development and Cell Proliferation Mol. Cell. Biol., July 1, 2004; 24(13): 5863 - 5874. [Abstract] [Full Text] [PDF]
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V. Vulsteke, M. Beullens, A. Boudrez, S. Keppens, A. Van Eynde, M. H. Rider, W. Stalmans, and M. Bollen Inhibition of Spliceosome Assembly by the Cell Cycle-regulated Protein Kinase MELK and Involvement of Splicing Factor NIPP1 J. Biol. Chem., March 5, 2004; 279(10): 8642 - 8647. [Abstract] [Full Text] [PDF]
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 H. T. Tran, A. Ulke, N. Morrice, C. J. Johannes, and G. B. G. Moorhead Proteomic Characterization of Protein Phosphatase Complexes of the Mammalian Nucleus Mol. Cell. Proteomics, March 1, 2004; 3(3): 257 - 265. [Abstract] [Full Text] [PDF]
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 H. CEULEMANS and M. BOLLEN Functional Diversity of Protein Phosphatase-1, a Cellular Economizer and Reset Button Physiol Rev, January 1, 2004; 84(1): 1 - 39. [Abstract] [Full Text] [PDF]
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T. Ammosova, M. Jerebtsova, M. Beullens, Y. Voloshin, P. E. Ray, A. Kumar, M. Bollen, and S. Nekhai Nuclear Protein Phosphatase-1 Regulates HIV-1 Transcription J. Biol. Chem., August 22, 2003; 278(34): 32189 - 32194. [Abstract] [Full Text] [PDF]
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Q. Jin, A. van Eynde, M. Beullens, N. Roy, G. Thiel, W. Stalmans, and M. Bollen The Protein Phosphatase-1 (PP1) Regulator, Nuclear Inhibitor of PP1 (NIPP1), Interacts with the Polycomb Group Protein, Embryonic Ectoderm Development (EED), and Functions as a Transcriptional Repressor J. Biol. Chem., August 15, 2003; 278(33): 30677 - 30685. [Abstract] [Full Text] [PDF]
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