From the Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520
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ABSTRACT |
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Protein tyrosine phosphatases are involved in the regulation of important cellular processes such as signal transduction, cell cycle progression, and tumor suppression. Here we report the cloning and characterization of PIR1, a novel member in the dual-specificity phosphatase subfamily of the protein tyrosine phosphatases. PIR1 also contains two stretches of arginine-rich sequences. We have shown that the recombinant PIR1 protein possessed an intrinsic phosphatase activity on phosphotyrosine-containing substrate. A unique feature of this phosphatase is that it binds directly to RNA in vitro with high affinity. In addition, we have found that PIR1 interacted with splicing factors 9G8 and SRp30C, possibly through an RNA intermediate during a yeast two-hybrid screen. PIR1 exhibited a nuclear-staining pattern that was sensitive to RNase A, but not to DNase I, suggesting that PIR1 in the cells are associated with RNA and/or ribonucleoprotein particles. Furthermore, a fraction of PIR1 showed a speckle-staining pattern that superimposed with that of the splicing factor, SC35. Taken together, our data suggest that PIR1 is a novel phosphatase that may participate in nuclear mRNA metabolism.
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INTRODUCTION |
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Protein tyrosine phosphatases, in conjunction with protein tyrosine kinases, regulate the levels of protein tyrosine phosphorylation important for cell growth, differentiation, or transformation (1, 2). Protein tyrosine phosphatases (PTP)1 can be grouped as classic PTPs (including receptor-like PTPs and cytoplasmic PTPs), dual-specificity phosphatases, and low molecular weight (acid) phosphatases (2). Both classic PTPs and dual-specificity phosphatases contain a conserved signature motif (HCXXGXXRXG), which constitutes the active site in the phosphatase catalytic domain (3). The conserved cysteinyl residue in this motif is required for the formation of a thiophosphate intermediate during the phosphate transfer reaction (3).
Dual-specificity phosphatases, a subfamily of protein tyrosine
phosphatases, play important roles in signal transduction, cell cycle
regulation, and tumor suppression. Although dual-specificity phosphatases contain little primary sequence homology to classic PTPs,
they share a similar structural folding, especially at the catalytic
site, with classic PTPs (3). Some members of this subfamily of enzymes
have been shown to be able to dephosphorylate both phosphotyrosine and
phosphoserine/phosphothreonine. One well known member of the
dual-specificity phosphatases is MKP-1/CL100, a highly selective
phosphatase that dephosphorylates and inactivates mitogen-activated
protein kinases (4, 5). Another example is Cdc25 (6), which
dephosphorylates the inhibitory phosphotyrosine and phosphothreonine
residues in Cdc2, a cyclin-dependent kinase required for
G2 to M phase transition during cell cycle progression. More recently, PTEN/MMAC1/TEP1, a novel phosphatase that is encoded by
a tumor suppressor locus on chromosome 10q23 and its mRNA level is
regulated by transforming growth factor (7-10), has been added to
this subfamily of protein tyrosine phosphatases.
Here we report the cloning and characterization of a novel phosphatase that structurally belongs to the dual-specificity phosphatase subfamily. Interestingly, this novel enzyme can bind to RNA in vitro, and associate with RNA and/or ribonucleoprotein (RNP) complexes in vivo. We have therefore named this novel enzyme PIR1, as phosphatase that interacts with RNA/RNP complex 1.
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MATERIALS AND METHODS |
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cDNA Cloning--
The ML1 ZAPII cDNA library (9) was
screened using the EST clone H60626 as a probe. Twenty positive clones
were identified and excised with ExAssist helper phage (Stratagene). By
restriction mapping, clone 14 was shown to contain the longest insert.
This clone was subjected to DNA sequencing on both strands. The region containing the putative open reading frame was amplified by the polymerase chain reaction (PCR) with a BamHI site added at
the ends of both primers. The 1-kilobase BamHI fragment was
subcloned into pCGT (11), pGBT9 (CLONTECH), and
pAcG1 (PharMingen) and sequenced again. Site-directed mutagenesis was
carried out to change cysteine 152 to serine (C
S) using the
QuikChange method (Stratagene). The presence of the mutation was
confirmed by DNA sequencing. T epitope-tagged PIR1 or PIR1(CS) was
amplified from pCGT-PIR1 or pCGT-PIR1(CS) template by PCR with
BglII linker added at the ends of the primers and subcloned
into pVL1393 (PharMingen).
Cell Lines, Northern Blot, and Western Blot Analyses-- Cells culture and Northern blot analysis were performed as described previously (9). For Western blot analysis, cell lysates were resolved on 10% SDS-PAGE, and proteins were blotted onto nitrocellulose membrane. Anti-T epitope antibody (Novagen) and 9E10 (Oncogene) were applied in TTBS (20 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween 20) with 5% nonfat milk. Blots were then washed extensively with TTBS and then incubated with appropriate secondary antibody conjugated with horseradish peroxidase. The immunoreactive proteins were detected with enhanced chemiluminescence (ECL; NEN Life Science Products).
Recombinant Protein Expression-- Recombinant baculoviruses were obtained by cotransfecting plasmid pVL1393-T-PIR1 (WT or CS), pAcG1-PIR1 (WT or CS) or pAcG1-MKP1-myc with linear baculovirus DNA (PharMingen) into Sf9 cells according to the standard protocol of PharMingen. Sf9 cells were infected with the recombinant baculoviruses. 72 h post-infection, cells were harvested, resuspended in Buffer A (20 mM Tris, pH 7.4, 10 mM KCl, 1.5 mM MgCl2, 5 mM dithiothreitol, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM benzamidine) and lysed by Dounce homogenization. Lysates were centrifuged at 13,000 rpm for 20 min to remove the insoluble cell debris. The supernatant was then supplemented with either 100 mM KCl (for single-stranded DNA binding assay) or 140 mM KCl (for phosphatase activity assay) and 0.1% Tween 20.
Protein Tyrosine Phosphatase Activity Assay-- The clarified Sf9 cell lysates containing glutathione S-transferase(GST)-PIR1 or GST-PIR1(CS) fusion protein (1 ml) were incubated with 100 µl of glutathione-Sepharose beads (Amersham Pharmacia Biotech). After 2 h incubation at 4 °C, beads were collected and washed extensively with buffer A plus 140 mM KCl, and then assayed for phosphatase activity using phosphotyrosyl-containing poly(Glu4Tyr1) as substrate. Poly(Glu4Tyr1) (Sigma) was phosphorylated with the kinase domain of the insulin receptor (BIRK), and the protein tyrosine phosphatase activity assays were carried out as described previously (9).
Single-stranded DNA Binding Assay-- Cell extracts (400 µl) were incubated with 150 µl single-stranded DNA agarose (Life Technologies, Inc.) in buffer B (20 mM Tris, pH 7.4, 100 mM KCl, 5 mM dithiothreitol, 0.1% Tween 20, 10 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM benzamidine) for 2 h at 4 °C. The beads were washed 4 times with 1 ml of buffer B, and the proteins were then eluted sequentially with 400 µl each of 0.25 M, 0.5 M, 1 M NaCl in 20 mM Tris buffer (pH 7.4). The proteins retained on the beads after 1 M NaCl elution were released by boiling in 800 µl of Laemmli sample buffer. Equal volumes of input, flow-through, eluates at different salts, and bound proteins at 1 M salt were analyzed by SDS-PAGE and Western blot analysis.
Southwestern and Northwestern Blot
Analyses--
Baculovirus-expressed GST-PIR1 (WT or CS) or GST-MKP-1
were affinity purified by binding to glutathione-Sepharose beads as described above. The bound proteins were analyzed by 10% SDS-PAGE and
then transferred onto nitrocellulose filter. The filters were washed
with TTBS then phosphate-buffered saline. Proteins on the filter were
denatured with 6 M guanidine hydrochloride in TBB solution
(20 mM Tris pH 7.4, 5 mM MgCl2, and
75 mM KCl) and renatured by sequential incubation (10 min
each) in 3, 1.5, 0.75, 0.375, 0.187, and 0 M guanidine
hydrochloride in TBB solution. Filters were blocked with nucleic acid
binding buffer (Tris pH 7.5, 1 mM EDTA, 50 mM
NaCl, and 1× Denhardt's solution) for 30 min. The filters were
incubated with the radiolabeled DNA or RNA probes for 1 h at room
temperature, then washed with the nucleic acid binding buffer (three
times, 5 min each) before autoradiography. A single-stranded DNA probe
was prepared with [-32P]dATP using the random
primer-labeling kit (Stratagene) with sheared single-stranded salmon
sperm DNA as the template. To compare the binding preference of PIR1
for single-stranded DNA or RNA by Southwestern and Northwestern blot
analyses, cDNAs encoding PTEN/MMAC1/TEP1, cyclin A,
p45SKP2, p27KIP1, and p21WAF1 were
each PCR amplified with T3 and T7 primers from the corresponding pBluescript-based plasmid. The PCR products were gel-purified, pooled,
and used as templates. The 32P-labeled DNA probe was
prepared by asymmetric PCR using Taq DNA polymerase, T3
primer, and [
-32P]dATP for 40 cycles. The
32P-labeled RNA probe was generated with T3 RNA polymerase
using [
-32P]ATP and the Riboprobe kit (Promega) for
1 h at 30 °C. Both [
-32P]dATP and
[
-32P]ATP were used at 4 × 105
cpm/pmol in the labeling reactions. The DNA and RNA probes were each
purified with G25 sizing column (Boehringer Mannheim) and adjusted to
106 cpm/ml for filter binding assays.
Yeast Two-hybrid Screen--
YRG2 (Stratagene) cells were
transformed with 400 µg of HeLa pGADGH library DNA (12) and 400 µg
of pGBT9-PIR1 DNA. Transformants were selected for histidine, leucine,
and tryptophan prototrophs according to Stratagene's protocol.
Histidine prototroph colonies (His+) were then tested by
the -galactosidase assay. About 100 His+
-galactosidase+ colonies were obtained,
and 20 of them were further analyzed. Plasmids were recovered from the
yeast cells and the pGADGH constructs were selected in the
Escherichia coli MH4 strain (12) based on their ability to
confer leucine prototroph. pGADGH plasmids were then re-transformed
into YRG2 strain together with either pGBT9-PIR1 or pGBT9-CDK6 plasmid.
Plasmids encoding proteins that showed specific interaction with PIR1
but not with CDK6 were sequenced. The positive cDNAs that were
fused in-frame with the upstream Gal4 activation domain include genes
encoding splicing factor 9G8 or SRp30C.
Immunostaining-- HeLa cells were transfected with 1 µg of pCGT-PIR1 and 19 µg of pUC18 carrier DNA by the calcium phosphate method. Cells were washed with phosphate-buffered saline 36 h post-transfection and permeabilized with CSK buffer (10 mM Pipes, pH 6.8, 300 mM sucrose, 3 mM MgCl2, 1 mM EGTA, 0.5% Triton X-100, 100 mM NaCl, 1 mM phenylmethylsulfonyl fluoride) (13). For the nuclease sensitivity experiments, cells were incubated at 27 °C for 1 h with CSK buffer supplemented with either (i) 400 units/ml DNase I, 20 mM vanadyl-ribonucleoside complex and 50 mM NaCl; (ii) 0.1 mg/ml RNase A; or (iii) 20 mM vanadyl-ribonucleoside complex and 50 mM NaCl (control). The cells were then extracted with 250 mM (NH4)2SO4 in CSK buffer at room temperature for 10 min and fixed with 3.7% formaldehyde in CSK buffer at 4 °C for 30 min. Cells were stained with anti-T epitope antibody (Novagen), followed by rhodamine-conjugated donkey antibody to mouse IgG (Jackson ImmunoResearch Laboratories). For immunocolocalization of PIR1 and SC35, cells were permeabilized in CSK buffer in the absence of RNase A inhibitor (vanadyl-ribonucleoside complex) at 27 °C for 1 h, extracted with 250 mM (NH4)2SO4 in CSK buffer at room temperature for 10 min and then fixed with 3.7% formaldehyde in CSK buffer. Cells were stained with monoclonal antibody SC35 (ATCC), followed by rhodamine-conjugated donkey antibody to mouse IgG. The coverslips were then incubated with biotinylated anti-T epitope antibody (Novagen) followed by fluorescein-conjugated streptavidin (Jackson ImmunoResearch Laboratories). Cells were examined with a Bio-Rad confocal microscope MRC-600 and the imaging data was processed with computer program Adobe Photoshop 4.0.
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RESULTS |
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Cloning of a Novel Member of Dual-specificity Phosphatases-- To identify novel dual-specificity phosphatases, we have used primers corresponding to the conserved sequences HCTHGIN and FEQARGH in the catalytic domain of several dual-specificity phosphatases to amplify gene sequences from human cDNA libraries by polymerase chain reaction. In addition, we have used these sequences to directly search the GenBankTM EST data base. These combined approaches have led us to identify a partial cDNA (EST clone H60626) that potentially encoded a novel phosphatase. To obtain its full-length cDNA, we used the EST clone H60626 as a probe to screen a cDNA library constructed from ML1 cells (a human myeloid cell line). The full-length cDNA sequence consists of 1593 nucleotides that potentially encode a protein of 329 amino acids (Fig. 1A). We have named this novel phosphatase PIR1 (phosphatase that interacts with RNA/RNP complex 1).
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PIR1 Possesses Intrinsic Protein Tyrosine Phosphatase Activity-- Although PIR1 carries the signature motif of protein tyrosine phosphatase, whether it bears protein phosphatase activity needs to be directly demonstrated. To do so, we have expressed PIR1 in insect Sf9 cells using the recombinant baculoviruses because the PIR1 protein is not stable in E. coli. We have expressed a fusion protein in which PIR1 was fused at the C terminus of GST (GST-PIR1). GST-PIR1 were affinity-purified by binding to glutathione-Sepharose beads. GST-PIR1 displayed protein tyrosine phosphatase activity toward tyrosyl-phosphorylated poly(GluTyr) (Fig. 3). Poly(GluTyr) is a random polymer of glutamate and tyrosine, and the tyrosyl-phosphorylated poly(GluTyr) has been shown to be an excellent in vitro substrate for dual-specificity phosphatases such as PTEN/MMAC1/TEP1 (10). As control, we have assayed in parallel the PIR1 derivative carrying the cysteine 152 to serine mutation (C152S) in the tyrosine phosphatase signature motif. The tyrosine phosphatase activity of GST-PIR1 was abolished by the C152S mutation (Fig. 3). These studies suggest that PIR1 possesses an intrinsic protein tyrosine phosphatase activity. Under similar conditions, we could not detect phosphatase activity using phosphoseryl/threonyl casein as substrate (data not shown). This could be because of the substrate selectivity of PIR1. It remains possible that in vivo PIR1 may dephosphorylate phosphoseryl/threonyl residues in addition to phosphotyrosyl residues with its physiological substrates.
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PIR1 Interacts Directly with Single-stranded DNA or RNA in Vitro-- PIR1 contains two stretches of arginine-rich regions (Fig. 1A). Because the arginine-rich regions in the viral protein Rev and Tat are known to be involved in binding to RNA (16), we tested whether PIR1 can interact with RNA. Because many RNA-binding proteins can bind to both RNA and single-stranded DNA in vitro, we first tested whether PIR1 can bind to single-stranded DNA. Wild type PIR1 or PIR1(CS) mutant were expressed as T epitope-tagged forms by recombinant baculoviruses in Sf9 cells. When the cell lysates from such baculovirus-infected Sf9 cells were incubated with single-stranded DNA immobilized on agarose beads, most of the PIR1 or PIR1(CS) proteins were retained on the beads (Fig. 4A). The binding of PIR1 or PIR1(CS) to single-stranded DNA beads was resistant to a 0.25 M salt wash, and even after a 1 M salt wash, a substantial fraction of the proteins were still retained on the beads (Fig. 4A). The relative resistance of PIR1-DNA interactions to medium salt washes suggests that PIR1 or its CS mutant protein can bind to single-stranded DNA with high affinity.
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PIR1 Interacts with RNPs in the Yeast Two-hybrid System--
To
gain an insight into the cellular processes that are regulated by PIR1,
we have employed the yeast two-hybrid screen method to identify
proteins that may interact with PIR1. By screening the HeLa cDNA
yeast two-hybrid library with PIR1 as a bait, we have identified ~100
positive clones in both histidine prototroph and -galactosidase
assay, and 20 of them were chosen for further analysis. These plasmids
were recovered from yeast colonies. To test the specificity of the
interaction with the bait protein, each plasmid was then retransformed
into the yeast strain together with a bait plasmid encoding either PIR1
or CDK6. The plasmids that conferred interactions only with PIR1, but
not with CDK6 control, were then sequenced. Two of the cDNA clones
were found to encode splicing factors 9G8 or SRp30C, both as an
in-frame fusion with the upstream Gal4 transcription activation domain (Fig. 5). Both 9G8 and SRp30C belong to
the SR family splicing factors, which share a domain containing serine
and arginine repeats and are components of the mRNA-splicing
complexes (21). So far, we have not been able to detect physical
association between PIR1 and splicing factors in mammalian cells. It is
possible that the interaction of PIR1 with splicing factors 9G8 or
SRp30C is mediated through an RNA intermediate, as PIR1 itself can bind
to RNA directly, and our coimmunoprecipitation method may not be
sensitive enough to detect such interaction.
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Immunolocalization of the Ectopically Expressed PIR1-- To examine the cellular localization of PIR1, we expressed the T epitope-tagged PIR1 in HeLa cells by the transient transfection method, followed by immunofluorescence staining with anti-T epitope antibody. PIR1 was localized to the nuclei with the exclusion of the nucleolus, and no staining was observed when cells were transfected with the vector alone (Fig. 6, A and B). To determine whether PIR1 in cells is associated with certain nucleic acids or protein complexes involved in nucleic acids metabolism, we subjected the permeabilized cells to DNase I or RNase A treatment before fixation. Although PIR1 staining was not affected by pretreatment with DNase I, it was greatly diminished by pretreatment with RNase A (Fig. 6, C and D). The sensitivity of PIR1 staining to RNase A suggests that PIR1 in mammalian cells is associated with RNA and/or RNP complexes involved in nuclear mRNA metabolism.
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DISCUSSION |
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Our studies demonstrate that PIR1 is a novel member of the dual-specificity subfamily of protein tyrosine phosphatases that exhibits high affinity to RNA. PIR1 bound directly to the in vitro transcribed RNAs derived from cDNA templates. Using PIR1 as a bait, we have isolated cDNA clones encoding accessory splicing factors 9G8 and SRp30C that showed specific interactions with PIR1 in the yeast two-hybrid system. When ectopically expressed in HeLa cells, PIR1 manifested a nuclear staining pattern, and the staining was removed by pretreatment of cells with RNase A but not DNase I. Furthermore, a fraction of PIR1 was colocalized with the splicing factor SC35 in speckles. PIR1 is the first member in the protein tyrosine phosphatase family that shows high affinity for RNA both in vitro and in vivo. Our studies suggest that PIR1 may participate in nuclear mRNA metabolism.
Increasing evidence suggests the participation of protein kinases and phosphatases in mRNA processing (21, 23). For example, a dual-specificity kinase Clk/Sty is found to be a partner with SR splicing factors in a yeast two-hybrid screen, and a catalytic inactive form of this kinase is colocalized with SR splicing factors in speckles in transfected cells (24). Serine/threonine phosphatase 1, or a serine/threonine phosphatase-1-like activity, has been shown to affect the subnuclear localization of the splicing factors (25). More recently, CEL-1, a phosphatase in C. elegans was shown to be a 5' triphosphatase for RNA molecules and was suggested to be involved in the mRNA capping reaction (15). Whether PIR1 is involved in regulating pre-mRNA splicing or other aspects of mRNA metabolism such as capping, polyadenylation, stability, or transport awaits further studies.
One interesting question arising from our studies is which region or domain in PIR1 mediates its interaction with RNA. Our observations that both the wild type PIR1 and its catalytically inactive C152S mutant bind to RNA with comparable affinity, and our data that binding can take place with denatured phosphatases raises the interesting possibility that interaction of PIR1 with RNA may not require the catalytic center nor the native conformation of the enzyme. One possibility is that PIR1 binds to RNA through its arginine-rich sequences. Several specific RNA binding proteins, such as Rev and Tat, are known to contain an arginine-rich motif (16). Rev, a protein encoded by HIV, can bind and facilitate nuclear export of intron-containing viral RNA. Tat, also encoded by HIV, is involved in the regulation of transcription by binding to viral mRNA. Very little identity is found between the arginine-rich motif sequences except for the richness of arginine residues. Further studies are required to determine whether the arginine-rich regions in PIR1 mediate its binding to RNA.
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ACKNOWLEDGEMENTS |
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We would like to thank Drs. Susan Baserga and Hui Zhang for helpful discussions and critical reading of the manuscript, Peer Bork and Anne Marie Quinn for assistance with the computer sequence analysis.
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FOOTNOTES |
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* This work was supported by the Donaghue Medical Research Foundation.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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AF023917.
Recipient of the Leslie H. Warner Fellowship in Cancer
Research.
§ Pew Scholar in the Biomedical Sciences. To whom correspondence should be addressed: Dept. of Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06520. Tel.: 203-737-1923; Fax: 203-785-7023; E-mail: hong.sun{at}yale.edu.
The abbreviations used are: PTP, protein tyrosine phosphatase(s); RNP, ribonucleoprotein; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; WT, wild type; GST, glutathione S-transferasePipes, 1,4-piperazinediethanesulfonic acid.
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REFERENCES |
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