(Received for publication, January 23, 1997, and in revised form, May 21, 1997)
From Cold Spring Harbor Laboratory, Cold Spring
Harbor, New York 11724-2208 and
Northwestern University Medical
School, Chicago, Illinois 60611
Alternative splicing of the T-cell protein
tyrosine phosphatase (TCPTP) transcript generates two forms of the
enzyme that differ at their extreme C termini: a 48-kDa endoplasmic
reticulum-associated form and a 45-kDa nuclear form. By affinity
chromatography, using GST-TCPTP fusion proteins, we have isolated three
cytoplasmic proteins of 120, 116, and 97 kDa that interact with TCPTP.
The p120 protein associated with residues 377-415 from the C terminus of the 48-kDa form of TCPTP, whereas the recognition site for p97 and
p116 was mapped to residues 350-381 encompassing the TCPTP nuclear
localization sequence (NLS). The TCPTP NLS was shown to be bipartite,
requiring basic residues 350-358 (basic cluster I) and 377-381 (basic
cluster II), the sites of interaction with p97 and p116, for efficient
nuclear translocation. The interaction between p97, p116, and the TCPTP
NLS appeared unique in that these proteins did not form a stable
interaction with the classical NLS of SV40 large T antigen or the
standard bipartite NLS of nucleoplasmin. Sequence analysis of p97
identified it as the nuclear import factor p97 (importin-), which is
an essential component of the nuclear import machinery. In assays
in vitro in permeabilized cells, p97 was necessary but not
sufficient for optimal nuclear import of TCPTP. We found that TCPTP
co-immunoprecipitated with the nuclear import factor p97 from cell
lysates and that purified recombinant p97 and TCPTP interacted directly
in vitro. These results indicate selectivity in the binding
of p97 and p116 to the TCPTP NLS and suggest that p97 may mediate
events that are distinct from the classical nuclear import process.
Moreover, these results demonstrate that the C-terminal segment of
TCPTP contains docking sites for interaction with proteins that may
function to target the enzyme to defined intracellular locations and in
the process regulate TCPTP function.
Protein tyrosine phosphorylation is an essential element in the control of fundamental cellular signaling events involved in proliferation and differentiation. The level of cellular tyrosine phosphorylation is determined by the activity of both protein-tyrosine kinases and protein tyrosine phosphatases (PTPs)1 (1, 2). Structurally, the PTPs are characterized by a conserved catalytic domain of approximately 240 residues and N- or C-terminal noncatalytic segments that frequently serve regulatory functions (1, 2). Like the protein tyrosine kinases, the PTPs can be subdivided into transmembrane and nontransmembrane forms (1, 2). The function of the PTPs can be modulated by interaction between the noncatalytic segment of these enzymes and various binding proteins (3). Protein-protein interactions have the potential to modulate PTP activity either by altering enzyme activity directly or by controlling intracellular localization.
The cDNA encoding the T-cell PTP (TCPTP) was originally isolated from a human peripheral T-cell library (4). This cDNA encodes a 48-kDa protein that displays 65% sequence identity overall with PTP1B and 74% sequence identity within the conserved catalytic domain. This enzyme is ubiquitously expressed in human tissues and has a modular structure comprising an N-terminal catalytic domain and a noncatalytic C-terminal segment. The 48-kDa TCPTP is primarily localized to the particulate fraction of cell extracts and requires treatment with detergent for extraction (5, 6). In contrast, a 37-kDa protein comprising the TCPTP catalytic domain and lacking the C-terminal segment is soluble (5, 6), suggesting that the C-terminal segment is important for localization. Indeed a stretch of 19 hydrophobic residues at the extreme C terminus of TCPTP have been shown to be responsible for targeting the 48-kDa TCPTP to the ER (7). Furthermore, in gel filtration experiments the expressed 48-kDa TCPTP migrates as a high molecular weight complex of >600 kDa (5), whereas the 37-kDa protein migrates at the expected molecular weight (5). These results suggest that the C-terminal segment of TCPTP may interact with other proteins and that such interacting proteins may have targeting functions.
Alternative splicing of TCPTP message gives rise to a 45-kDa form that
lacks the hydrophobic segment at the extreme C terminus (residues
382-418) (8-10) (Fig. 1A). This 45-kDa form of TCPTP is
found in the nucleus, and a nuclear localization sequence (NLS) has
been identified in the C-terminal segment (residues 350-381) (7, 10).
As such, the 48- and 45-kDa forms of TCPTP are targeted to two distinct
sites, the ER and the nucleus, respectively (7). It seems probable that
discrete protein-protein interactions involving the C-terminal segments
of the two TCPTP variants may control their intracellular location. As
a first step in understanding TCPTP function we have concentrated on
identifying proteins that bind to the targeting segment of the
phosphatase. In this study we have characterized three
TCPTP-interacting proteins: the nuclear import factor p97
(importin-) and two novel proteins, p116 and p120, that interact
with distinct as well as overlapping sites within the TCPTP C
terminus.
pGEX-KG constructs encoding human TCPTP
truncation mutants were generated by PCR using the cloned
Pfu DNA polymerase (Stratagene, La Jolla, CA).
Oligonucleotides incorporated a BamHI site immediately 5 to
the initiation codon and an EcoRI site immediately 3
to the
termination codon. The 5
oligonucleotide used for generating truncation mutants of the full-length TCPTP (residues 1-415)
was 5
-GGCTCCCGGATCCATGCCCACCACCATCGAGCGGGAG-3
, whereas that used for
generating truncation mutants of the TCPTP C-terminal segment (residues
318-415) was 5
-GAATGGGGGATCCATAGGTCTAGAAGAAGAAAAACTG-3
. 3
oligonucleotides used for generating TCPTP truncation mutants included
an in frame stop codon prior to the EcoRI site (TC-(1-415), 3
-GACAAAAAAGTCGTTTTACGGGATATTCTTAAGTTAAAAG-5
; TC-(1-381) and TC-(318-381), 3
-CTCTTACTTGCTTTTTCTTTTTCCATTCTTAAGACCGTTC-5
; TC-(1-349) and TC-(318-349),
3
-CTCCTCTTGTCACTCTCACGAGATATTCTTAAGTAAGCTC-5
). cDNAs encoding the
various TCPTP truncation mutants were subcloned into BamHI
and EcoRI sites in the Escherichia coli
expression vector pGEX-KG to generate in frame glutathione
S-transferase (GST) fusions. The pGEX TCPTP construct
encoding residues 1-317 was a kind gift from Dr. D. Barford (Oxford
University). pET-21a(+) (Novagen, Madison, WI) constructs encoding GST
alone (pET-GST) or a GST fusion of the 48-kDa form of TCPTP
(pET-GST-TCPTP48) were kind gifts from Drs. H. Charbonneau and Luning
Hao (Purdue University, West Lafayette, IN). Recombinant pUC18
constructs encoding the human 45- and 48-kDa forms of TCPTP and the
inactive 45-kDa C216S TCPTP mutant were a kind gift from Drs. J. A. Lorenzen and E. H. Fischer (University of Washington, Seattle,
WA). The pUC18 TCPTP constructs were digested with EcoRI and
HindIII, and cDNA fragments were subcloned into the
pBluescriptR II KS (+) vector (Stratagene, La Jolla, CA).
pET-21a(+) TCPTP NLS deletion mutants were generated by
oligonucleotide-directed mutagenesis (
350-352,
GAGGAGAACAGTGAGAGTGCTCTAATTCGAGAGGACAGAAAG;
350-358,
GAGGAGAACAGTGAGAGTGCTCTAGCCACCACAGCTCAGAAGGTG;
377-381, CAGAGGCTAAATGAGAATGAATGGTTATATTGGCAACCTATTC) of the human 48-kDa TCPTP DNA in pBluescriptR II KS (+) and NcoI
(internal site)/XhoI cDNA fragments exchanged with that
of the pET-GST-TCPTP48 construct. The pGEX construct encoding
nucleoplasmin (NPL) residues 88-200 was generated by subcloning the
NcoI/EcoRI fragment from Bluescript-NPL
(generously provided by Dr. Colin Dingwall, State University of New
York at Stony Brook) into the SmaI/EcoRI sites of
pGEX-KG after blunt ending the NcoI site.
pcDNA3 (Invitrogen, San Diego, CA) constructs encoding the wild
type 45-kDa TCPTP or the inactive C216S mutant were generated by
subcloning BamHI/XhoI fragments from the
respective TCPTP-Bluescript constructs. pSVL (Pharmacia, Uppsala,
Sweden) constructs encoding the human 45- and 48-kDa forms of TCPTP
were a kind gift from Drs. J. A. Lorenzen and E. H. Fischer
(University of Washington, Seattle, WA) (7). pSVL constructs encoding
TCPTP truncation mutants were generated by PCR. The 5 oligonucleotide
used in PCR reactions corresponded to an internal XbaI site
(5
-GAATAGGTCTAGAAGAAGAAAAACTGACAG-3
). The 3
oligonucleotides used to
generate the truncation constructs incorporated an in frame stop
codon followed by a BamHI restriction site (TC-(1-376),
3
-CTTTGTCTCCGATTTACTCTTACTTATTCCTAGGTTTTCCACC-5
; TC-(1-359),
3
-GCATAAGCTCTCCTGTCTTTCCGGATTCCTAGGGTCTTCGGG-5
; TC-(1-349),
3
-CTCCTCTTGTCACTCTCACGAGATATTCCTAGGTAAGCTCTC-5
). XbaI/BamHI-digested PCR products were
cloned into the XbaI/BamHI sites of the 45-kDa
TCPTP pSVL construct. pSVL constructs encoding the 45-kDa form of TCPTP
with residues 377-381 deleted (p45TC
RKRKR) or
residues 350-352 mutated to glutamine
(p45TCR350Q/K351Q/R352Q) were a kind gift from Drs. J. A. Lorenzen and E. H. Fischer (University of Washington, Seattle,
WA) (7). The pSVL construct encoding the 45-kDa form of TCPTP with
residues 377-381 deleted as well as residues 350-352 mutated to
glutamine was generated by PCR as described above for the pSVL
truncation mutants using the p45TCR350Q/K351Q/R352Q
construct as the template. The 3
oligonucleotide used to prime the
reaction was
3
-CTTGTCTCCGATTTACTCTTACTTGGTTCTAACTGTCTGTGGATTCCTAGGTACTGAACC-5
. The structures of the recombinant plasmids generated were
confirmed by restriction endonuclease analysis and the sequence of
cloned DNA confirmed by sequencing.
TCPTP truncation
mutants were expressed as GST fusion proteins in the E. coli
strain DH5 using the expression vector pGEX-KG or in the E. coli strain BL21(DE3) using the expression plasmid pET-21a(+). GST
fusion proteins were purified by affinity chromatography using
glutathione-Sepharose 4B (Pharmacia). Histidine-tagged human p97 (11,
12) or the p97 truncation mutant p97-(1-847) (12) were expressed in
the E. coli strain BL21(DE3) using the expression vector
pET-30a(+) and purified by affinity chromatography using Ni2+-nitrolotriacetic acid-agarose (Qiagen, Hilden,
Germany). Histidine-tagged importin-
was a kind gift from Dr. Colin
Dingwall.
HeLa, NIH 3T3,
WI 38, and COS1 cells were maintained in Dulbecco's modified Eagle's
medium with 10% fetal bovine serum and antibiotics (penicillin and
streptomycin) at 37 °C and 5% CO2. For metabolic
labeling, cells were cultured on 10-cm plates to ~70% confluence,
rinsed with methionine-free Dulbecco's modified Eagle's medium, and
subsequently starved in methionine-free Dulbecco's modified Eagle's
medium supplemented with 5% fetal bovine serum for 1 h. Cells
were incubated with 0.5 mCi/10-cm plate of
L-[35S]methionine Express Protein Labeling
Mix (DuPont) for 8 h. Labeled cells were washed three times with
ice-cold phosphate-buffered saline and harvested in phosphate-buffered
saline (1000 × g, 5 min). Harvested cells were frozen
with liquid nitrogen and either stored at 70 °C or lysed and used
immediately.
Where indicated, [35S]Met-labeled HeLa cells (1 × 10-cm plate) were resuspended in 0.8 ml of lysis buffer containing Triton X-100 (50 mM Tris·HCl, pH 7.5, 5 mM EDTA, 1% (w/v) Triton X-100, 150 mM NaCl, 5 mM iodoacetic acid) plus protease inhibitors (5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 mM benzamidine, and 2 mM phenylmethylsulfonyl fluoride). Lysates were precleared with 0.1 ml of IgG Sorb (The Enzyme Center, Malden, MA) for 1 h at 4 °C, DTT was added to 1 mM, and the supernatants (12,000 × g, 5 min, 4 °C) were incubated with glutathione-Sepharose coupled to 7.5 µg of GST, GST-1B-(1-435), GST-1B-(1-321), GST-1B-(290-435), GST-TC-(1-415), or GST-TC-(318-415) or approximately 15 µg of GST-TC-(1-317) for 2 h at 4 °C. Sepharose beads were washed with lysis buffer, and precipitates were analyzed by SDS-PAGE and autoradiography.
In all other cases [35S]Met-labeled HeLa cells (1 × 10-cm plate) were lysed in 0.8 ml of hypotonic lysis buffer (20 mM Tris·HCl, pH 7.5, 5 mM KCl, 1.5 mM MgCl2, 5 mM iodoacetic acid) plus protease inhibitors. Cells were allowed to swell for 20 min at 4 °C and then homogenized with a Dounce homogenizer. DTT was added to 2 mM, and the lysates were centrifuged at 70,000 × g, 4 °C for 20 min. Supernatants were made to 150 mM NaCl and 0.5% (w/v) Triton X-100 and 0.4 ml of each supernatant was gently agitated for 3 h at 4 °C with 2.5 µl of glutathione-Sepharose containing approximately 3 µg of immobilized GST-TCPTP fusion protein. GST-TCPTP fusion proteins were standardized for the apparent full-length protein. Sepharose beads were then collected by centrifugation and washed with lysis buffer containing 150 mM NaCl and 0.5% Triton X-100. Precipitates were resolved by SDS-PAGE and visualized following autoradiography.
Cellular Fractionation Studies[35S]Met-labeled HeLa cells (2 × 10-cm plate) were resuspended in 1.2 ml of buffer A (20 mM Tris·HCl, pH 7.5, 5 mM KCl, 1.5 mM MgCl2, 2 mM EGTA, 5 mM iodoacetic acid, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 mM benzamidine, and 2 mM phenylmethylsulfonyl fluoride) and allowed to swell for 20 min at 4 °C. The volume of one-half of the cell suspension was made to 1.2 ml with buffer B (100 mM Tris·HCl, pH 7.6, 0.5 M sucrose, 10 mM EDTA, 5 mM iodoacetic acid, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 mM benzamidine, and 2 mM phenylmethylsulfonyl fluoride), Triton X-100 was added to a final concentration of 1.0% (w/v), and the lysate was homogenized with a Dounce homogenizer (20 strokes) to yield the Triton X-100 lysate. The other half of the cell suspension was also homogenized with a Dounce homogenizer (20 strokes), and an equal volume of buffer B was added. This cell lysate was subsequently centrifuged at 1000 × g, 4 °C for 5 min. The pellet was resuspended in 1.2 ml of a 1:1 mixture of buffers A and B, Triton X-100 was added to 0.5% (w/v), and the lysate was termed the nuclear fraction. The supernatant was centrifuged at 70,000 × g, for 10 min, and the subsequent supernatant was termed the high speed supernatant. The pellet was resuspended in 1.2 ml of a 1:1 mixture of buffers A and B, and Triton X-100 was added to 1.0% (w/v). The resuspended pellet was incubated on ice for 60 min and centrifuged at 70,000 × g for 10 min, and the supernatant was termed the membrane fraction. 250 µl of each fraction was mixed with 2.5 µl of glutathione-Sepharose containing ~2 µg of immobilized GST or GST-TCPTP fusion protein, and the mixture was agitated for 3 h at 4 °C. Sepharose beads were then collected by centrifugation and washed six times with buffer C (50 mM Tris·HCl, pH 8.0, 5 mM EDTA, 1% (w/v) Triton X-100, 150 mM NaCl, 2 mM DTT, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 µg/ml pepstatin A, 1 mM benzamidine, 2 mM phenylmethylsulfonyl fluoride). Precipitates were resolved by SDS-PAGE and visualized following autoradiography.
Isolation and Sequencing of TCPTP-interacting ProteinsFor preparative isolation of TCPTP-binding proteins, 2.5 × 109 HeLa cells were resuspended in 40 ml of hypotonic lysis buffer, incubated on ice for 20 min, and homogenized with a Dounce homogenizer. All subsequent procedures were at 4 °C. The lysate was centrifuged at 100,000 × g for 30 min, and the resulting supernatant was brought to 150 mM NaCl, 0.5% (w/v) Triton X-100 and 2 mM DTT and applied to ~400 µg of either GST-TC-(318-415) or GST-TC-(1-317) immobilized on 200 µl of glutathione-Sepharose beads. The slurry was agitated for 2 h, and the beads were collected and washed with hypotonic lysis buffer containing 150 mM NaCl, 0.5% (w/v) Triton X-100, and 2 mM DTT. Sepharose beads were then resuspended in sample buffer, and TCPTP-binding proteins were resolved by SDS-PAGE and visualized by staining with Coomassie Brilliant Blue G (chromatographically purified and recrystallized) (Sigma). Individual protein bands were excised and digested in situ with 500 ng of Achromobacter protease I (Wako, Richmond, VA) in 100 mM Tris·HCl, pH 9.0, and 0.1% (w/v) Tween 20 for 20 h at 30 °C. Digests were resolved by chromatography on reverse phase HPLC, and individual peptides were sequenced on an Applied Biosystems protein sequencer.
Co-immunoprecipitation AssayHeLa cells from 2 × 10-cm plates (70% confluent) were resuspended in 0.8 ml of lysis buffer (50 mM Tris·HCl, pH 8.0, 5 mM EDTA, 1% (w/v) Triton X-100, 400 mM NaCl, 5 mM iodoacetic acid) plus protease inhibitors and agitated at 4 °C for 20 min. Lysates were then diluted with 1 volume of the same buffer without NaCl and precleared with 0.1 ml of IgG Sorb for 1 h at 4 °C. Precleared lysates were centrifuged (12,000 × g, 5 min, 4 °C), and TCPTP or p97 was immunoprecipitated from supernatants with 20 µg of either the monoclonal anti-TCPTP CF4 (generously provided by Dr. David Hill, Calbiochem Oncogene Research Products, Cambridge, MA) or the anti-p97 mAb3E9 (11), respectively, for 5 h at 4 °C. Immune complexes were collected on Protein A-Sepharose CL-4B (Pharmacia), washed with lysis buffer containing 200 mM NaCl, resolved by SDS-PAGE and transferred onto Immobilon (Millipore, Bedford, MA). Immunoblots were probed with either anti-TCPTP CF4 antibody or anti-p97 mAb3E9 antibody at 1 µg/ml.
Precipitation of Purified p97 and Importin-Approximately 5 µg of either GST, GST-TCPTP deletion
mutants, or GST-TC-(318-415) were immobilized on 5 µl of
glutathione-Sepharose and agitated at 4 °C for periods ranging from
4 h to overnight as indicated with either 5 or 10 µg of purified
p97 or p97-(1-847) or with 10 or 20 µg of purified importin- in
0.5 ml of binding buffer (20 mM Tris, pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, and 1 mM DTT). Precipitates were washed five times with the same
buffer and analyzed by SDS-PAGE and Coomassie Blue staining.
COS1 cells were seeded onto glass coverslips at 2.5-3 × 105/60-mm plate 24 h prior to transfection. Cells were transfected (10 µg of plasmid DNA) by the calcium phosphate precipitation method and processed for immunofluorescence at 36-48 h post-transfection as described previously (7). Affinity-purified polyclonal anti-TCPTP antibody 6228 (obtained from J. A Lorenzen and E. H. Fischer, University of Washington, Seattle, WA) was applied at 0.8 µg/ml for 1 h.
NLS Binding Assay[35S]Met-labeled HeLa cells from 4 × 10-cm dishes were resuspended in 2 ml of hypotonic lysis buffer containing protease inhibitors, homogenized with a Dounce homogenizer, and lysate was clarified at 70,000 × g, 4 °C for 20 min. NaCl was added to the supernatant to a concentration of 150 mM and concentrated 10-fold using a Centricon 10 microconcentrator (Amicon, Beverly, MA) previously blocked with 5 mg/ml BSA in lysis buffer. Concentrated supernatant was agitated at 4 °C for 1 h with 100 µg of biotinylated BSA-SV40 NLS (CGGGPKKKRKVED) conjugate or glutathione-Sepharose coupled to 10 µg of either GST-TC-(318-381), or GST-NPL-(88-200). 50 µl of streptavidin-agarose (Sigma) was then added to the biotinylated conjugates, and the incubation of all samples continued for a further 1 h. Precipitates were collected by centrifugation, washed with lysis buffer containing 150 mM NaCl, resolved by SDS-PAGE, and analyzed by autoradiography. GST-fusion protein GST-TC-(318-349) lacking the TCPTP NLS and biotinylated BSA-mutant SV40 NLS (CGGGPKNKRKVED) conjugate were used as controls. SV40 NLS peptide (CGGGPKKKRKVED) and mutant SV40 NLS peptide (CGGGPKNKRKVED) were coupled to BSA with the heterobifunctional cross-linker Sulfo-SMCC (Pierce). BSA conjugates contained 20 peptide molecules/BSA molecule as estimated by electrophoretic mobility. Biotin was coupled to the BSA conjugates at a 4:1 molar ratio using Biotin-X-NHS, WS (Calbiochem).
In Vitro Nuclear Import AssaysMadin-Darby bovine kidney
cells were grown on glass coverslips and permeabilized with digitonin
as described previously (13, 14). Either SV40 large T antigen
NLS-allophycocyanin conjugate or recombinant 45-kDa TCPTP was used as a
substrate in import reactions. The SV40 large T antigen NLS peptide was
conjugated to allophycocyanin at a ratio of 5 mol/mol as described
previously (15). Recombinant 45-kDa TCPTP, expressed in E. coli using the pET21 expression vector (Novagen, Madison, WI) and
purified to homogeneity by ion exchange followed by gel filtration
chromatography, was a kind gift from Drs. H. Charbonneau and Luning Hao
(Purdue University, West Lafayette, IN). Import experiments were
performed in a 50-µl volume containing purified transport factors
(400 nM importin-, 400 nM p97, 1 µM Ran-GDP, and 1 µM RanBP1) and either 1 µg of the SV40 NLS-allophycocyanin substrate or 2 µg of the recombinant 45-kDa TCPTP as described previously (15).
TCPTP and PTP1B are closely related nontransmembrane
PTPs. Structurally they both comprise a catalytic domain linked at its C-terminal end to a noncatalytic, targeting segment (Fig.
1). To identify proteins that interact
with the C termini of these two enzymes, GST fusion proteins of the
C-terminal segments of TCPTP and PTP1B were expressed in E. coli and purified on glutathione-Sepharose beads. GST fusion
proteins of the catalytic domains of TCPTP and PTP1B were also produced
and used as controls. The immobilized fusion proteins were incubated
with Triton X-100 lysates of [35S]Met-labeled HeLa cells,
and associated proteins were precipitated (Fig.
2). The results indicated that, whereas
many proteins were retained by GST-PTP1B but not GST control, these
proteins all interacted with the catalytic domain of PTP1B
(GST-1B-(1-321)) and not with the C-terminal segment
(GST-1B-(290-435)). In contrast, GST-TCPTP interacted with three
proteins of 97 kDa (p97), 116 kDa (p116), and 120 kDa (p120) that were
precipitated by its C-terminal segment (GST-TC-(318-415)) but not by
its catalytic domain (GST-TC-(1-317)) (Fig. 2). Most striking was the
ability of TCPTP to precipitate sufficient quantities of p97 and p120
from ~5 × 106 HeLa cells for detection of the
proteins by Coomassie Blue staining of the gel, indicating that these
associated proteins readily formed highly stable complexes with the
TCPTP C terminus (data not shown). With the exception of material
derived from GST, no other proteins could be discerned in these
precipitates. We have concentrated on characterizing these
TCPTP-interacting proteins for two reasons. First, their primary site
of interaction appeared to be in the C-terminal segment of TCPTP, and
second, they appeared to be specific for the C terminus of TCPTP and
not PTP1B. Cellular fractionation of [35S]Met-labeled
HeLa cells and subsequent precipitation with the GST-TCPTP fusion
proteins illustrated that p97, p116, and p120 were present
predominantly in a high speed (70,000 × g) supernatant (Fig. 3), indicating that they are
primarily cytoplasmic.
Overlapping and Distinct Binding Sites of p97, p116, and p120 within the TCPTP C Terminus
The extreme C-terminal hydrophobic
residues of the 48-kDa form of TCPTP are essential for targeting to the
ER, whereas residues 350-381 encompass an NLS essential for the
nuclear import of the 45-kDa form (Fig. 1A) (7, 10). As
such, association of the TCPTP-interacting proteins with residues
within the TCPTP NLS or the hydrophobic C terminus would be consistent
with a role in targeting TCPTP to the ER or to the nucleus. To
characterize further the interactions of p97, p116, and p120 with the
TCPTP C terminus, a series of GST-fusion proteins with C-terminal
truncations in the 48-kDa TCPTP were generated and used to precipitate
interacting proteins from [35S]Met-labeled HeLa lysates.
In this case, lysates were prepared under hypotonic conditions, and
supernatants were subsequently brought to 0.5% (w/v) Triton X-100 and
150 mM NaCl to minimize nonspecific interactions. A GST
fusion protein of the full-length enzyme, GST-TC-(1-415), precipitated
p97 and p116 as well as p120 (Fig. 4,
A and B). Truncation of the full-length enzyme to
generate a mutant lacking the 34 C-terminal residues, GST-TC-(1-381),
resulted in loss of association with p120 but maintained the ability to bind to p116 and, albeit more weakly, to p97 (Fig. 4A). Upon
truncation of an additional 32 C-terminal residues, all binding to
these proteins was lost (Fig. 4A). Similar results were
obtained with lysates of NIH 3T3, WI38, and Jurkat cells (data not
shown), indicating that p97, p116, and p120 are not unique to HeLa
cells but are present both in transformed and nontransformed cell
lines. These results indicate that the C-terminal hydrophobic tail
(residues 382-415) of the 48-kDa form of TCPTP is necessary for the
interaction with p120, whereas p97 and p116 interact with residues
between 350 and 381.
Two clusters of basic amino acids (residues 350-358 and 377-381) can be delineated within the p97 and p116 binding segment. Previous studies have indicated that these basic clusters are important for nuclear import (7, 10). To characterize further the interaction between TCPTP and p97 and p116, GST-TCPTP mutants lacking residues 350-352 or 350-358 (basic cluster I) or 377-381 (basic cluster II) were generated, and their ability to precipitate proteins from [35S]Met-labeled HeLa lysates was investigated. We found that deletion of residues 350-352 (Fig. 4B) or 350-358 (data not shown) did not alter the association of p97 or p120 but did result in significant loss of p116 binding. In contrast, deletion of residues 377-381 (basic cluster II) resulted in loss of p97, p116, and p120 binding.
These results indicate that within the TCPTP NLS, the p97 binding site
includes residues in basic cluster II but not I, whereas the p116
binding site includes residues in both basic clusters. The absolute
dependence on an intact basic cluster II for p97 binding suggests that
the decreased association of p97 with the GST-TC-(1-381) protein
relative to GST-TC-(1-415) in Fig. 4A probably results from
the proteolysis of its C-terminal basic residues (377RKRKR381) rather than the loss of a binding
site in the hydrophobic C terminus. Consistent with this
interpretation, we find that a GST fusion protein of the TCPTP NLS is
sufficient to precipitate p97 and p116 from cell lysates (Fig.
5).
In addition, p120 binding requires an intact basic cluster II, and as such the p120 binding site can be extended to include basic cluster II (residues 377-381) as well as the hydrophobic C terminus (residues 382-415). However, basic cluster II alone is not sufficient for p120 association, since truncation of the C-terminal hydrophobic residues 382-415 led to a complete loss of p120 binding (Fig. 4A).
A Comparison of Proteins That Interact with TCPTP and Those That Bind to the NLS Motifs of SV40 Large T Antigen and NucleoplasminThe clusters of basic amino acids in the C-terminal segment of TCPTP that constitute the sites of interaction with p97 and p116 have been shown to be important for nuclear import of the phosphatase (7, 10). Therefore, we compared the abilities of the classical NLS of SV40 large T antigen (T-Ag), the standard bipartite NLS of nucleoplasmin, and the NLS of TCPTP to precipitate p97 and p116 from [35S]Met-labeled HeLa cell lysates. These experiments were performed at a variety of concentrations of cell lysate and under various detergent conditions. In all conditions tested, we found that although the TCPTP NLS (residues 350-381) precipitated p97 and p116, the SV40 T-Ag and nucleoplasmin NLS sequences did not (Fig. 5). These results highlight differences between protein-protein interactions involving the TCPTP NLS and the classical NLS sequences of the SV40 T-Ag and nucleoplasmin, raising the question of whether there are unique aspects to the mechanism of nuclear import of the phosphatase.
Isolation and Characterization of TCPTP-interacting ProteinsTo characterize further the TCPTP-interacting proteins,
preparative precipitations were undertaken using the C-terminal segment GST-TC-(318-415) immobilized on glutathione-Sepharose beads.
Precipitates were analyzed by SDS-PAGE, and TCPTP-interacting proteins
were visualized by Coomassie Blue staining. Two major proteins bound specifically to the TCPTP C-terminal domain (Fig.
6, inset). These migrated at
molecular weights corresponding to 97,000 (p97) and 120,000 (p120) as
expected from the results of the analytical scale experiments performed
with lysates of [35S]Met-labeled cells (Figs. 2 and 4).
The 116-kDa protein was also detected but at lower amounts than p97 and
p120. The p97 and p120 bands were excised and digested in
situ with Achromobacter protease I, and digests were
subsequently resolved by chromatography on reverse phase HPLC. The HPLC
profiles for p97 (Fig. 6) and p120 (data not shown) showed that these
proteins were unrelated and that p97 was not a proteolytic product of
p120. To date, the sequences of eight individual peptides from p120 do
not match any proteins in the data base. In contrast, the sequences of
two peptides from p97 (Fig. 6) were identical to sequences in the
nuclear import factor p97, otherwise known as karyopherin- or
importin-
(11). The nuclear import factor p97 is an essential factor
in the nuclear import of NLS-containing proteins (14). It localizes
both to the cytoplasm and to the nuclear envelope (11), consistent with our cellular fractionation experiments. Since the identity and function
of p116 and p120 are unknown, we have concentrated on characterizing
the association of TCPTP with p97.
Interactions of TCPTP with p97 in Vitro and in Vivo
Using the
NLS of the SV40 T-Ag as a model substrate, it has been demonstrated
that the 54/56-kDa NLS receptor (importin-) can bind directly to the
NLS, whereas p97 does not bind directly but enhances 54/56-kDa NLS
receptor binding (16-19). In light of this finding, we investigated
whether p97 interacted directly with TCPTP. We expressed p97 as a
histidine-tagged fusion protein and purified it on nickel-agarose.
Purified recombinant p97 was incubated subsequently with various
immobilized GST-TCPTP fusion proteins. We utilized the 48-kDa form of
TCPTP in these binding studies in vitro, because the
GST-45-kDa TCPTP is readily proteolyzed during its purification with
disruption of the p97 binding site (data not shown). The extent to
which p97 associated with the TCPTP truncation mutants was examined by
SDS-PAGE and Coomassie Blue staining (Fig.
7A). p97 was shown to bind
efficiently to the full-length 48-kDa TCPTP as well as to deletion
mutants lacking residues 350-352 or residues 350-358 (basic cluster
I) but not to a mutant lacking residues 377-381 (basic cluster II)
(Fig. 6A). The data illustrate that p97 can interact
directly with TCPTP independently of other proteins and that residues
377-381 are necessary for this interaction. These data are consistent
with the observed association between mutant TCPTP molecules and
proteins from lysates of [35S]Met-labeled HeLa cells
(Fig. 4).
To test whether TCPTP and p97 can associate at physiological levels of expression, the p97-specific antibody mAB3E9 and the TCPTP-specific antibody CF4 were used to test whether p97 and TCPTP co-immunoprecipitate from HeLa cell lysates. The 45-kDa form of TCPTP and low levels of the 48-kDa form were shown to co-immunoprecipitate with p97 from cell lysates (Fig. 7B). The ability of p97 to interact with low levels of the 48-kDa form of TCPTP indicates that part of the association may occur after lysis. We did not detect p97 in immunoprecipitates of TCPTP (data not shown); however, we have mapped the epitope for the TCPTP antibody CF4 to the last 38 residues of the 45-kDa form of the enzyme (data not shown), indicating overlap between the epitope and the p97 binding site. As such, steric hindrance due to antibody binding most likely prevented isolation of a complex with p97.
Comparison of the Interaction of TCPTP with p97/Importin-The classical mechanisms underlying nuclear
translocation of proteins depend upon interaction of importin- with
the protein to be imported. Considering that we did not detect
significant levels of importin-
associated with GST-TCPTP fusion
proteins following incubation of the phosphatase with labeled cell
lysates, we decided to compare directly the binding of importin-
with that of p97 to TCPTP using purified proteins (Fig.
8A). We observed that although
p97 formed a stable complex with the TCPTP C terminus, which could be
visualized by Coomassie Blue staining of SDS-PAGE gels of the
precipitates, the binding of importin-
was at best weak and at times
not detectable (Fig. 8A). In addition, we observed first
that TCPTP interacted with a truncated mutant of p97, p97-(1-847) (Fig. 8B), which does not bind importin-
(12) and,
second, that a 40-fold molar excess of the importin-
binding (IBB)
domain of importin-
(20, 21) did not prevent the association of p97
with TCPTP (data not shown). These results indicate that TCPTP does not
associate with p97 in the same way as importin-
and that the binding
site for TCPTP is unique. Moreover, these results first demonstrate the
strong association between p97 and TCPTP and, second, suggest that if
importin-
functions in the nuclear import of TCPTP this must reflect
a transient interaction. Moreover, since p97 formed a more stable
complex with the TCPTP NLS than importin-
, it is possible that
in vivo p97 may have a more direct role in nuclear transport
of TCPTP than previously described.
TCPTP Nuclear Localization in Transfected COS1 Cells
The
ability of p97 to interact directly with basic cluster II of the TCPTP
NLS suggests that it may function to target the 45-kDa form of TCPTP to
the nucleus. In overexpression studies, the 45-kDa form of TCPTP is
exclusively nuclear (7, 10) and its import is independent of
phosphatase activity (Fig.
9A). The contribution of basic
cluster II in nuclear import however is not clear. Lorenzen et
al. (7) have previously reported that residues 377-381 (basic
cluster II) constitute the TCPTP NLS. In contrast, Tillmann et
al. (10) have reported that residues 350-352 are sufficient to
target TCPTP to the nucleus. Because of the discrepancies as to the
exact nature of the TCPTP NLS, we have delineated further the TCPTP NLS
and investigated the relationship between p97 association (residues
377-381), p116 association (residues 350-352 and 377-381), and NLS
function. Truncation mutants encoding residues 1-376, 1-359, and
1-349 were expressed in COS1 cells, and their localization was
investigated by indirect immunofluorescence (Fig. 9B).
Although the nuclear translocation of all three truncated forms of
TCPTP was impaired relative to the full-length 45-kDa protein, the
localization of the 1-376 and 1-359 proteins was predominantly
nuclear. In contrast, the proportion of 1-349 protein in the cytosol
relative to the nucleus was higher than for either the 1-376 or 1-359
protein. The 1-349 protein appeared to be evenly distributed between
the nucleus and cytoplasm (Fig. 9B), and a similar
distribution was obtained for the catalytic domain (residues 1-317) of
TCPTP (data not shown). These results are consistent with the ability
of small proteins (40-60 kDa) to diffuse readily in and out of the
nucleus (reviewed by Dingwall and Laskey (22)). The results of this study indicate that although basic cluster I (residues 350-358) can
function to a limited extent to import TCPTP, basic cluster II
(residues 377-381) is also required for the efficient nuclear translocation of the enzyme. These results suggest that the TCPTP NLS
is bipartite in nature and help explain the discrepancies in previously
published work (7, 10).
In the work described by Lorenzen et al. (7), mutation of
basic residues 350-352 to glutamine
(p45TCR350Q/K351Q/R352Q) did not alter nuclear import,
whereas deletion of basic residues 377-381 (p45TCRKRKR)
impaired import. Nevertheless, the localization of the p45TC
RKRKR mutant appeared to be predominantly nuclear
(7). Localization similar to that for the p45TC
RKRKR
mutant was obtained in this study for the 1-376 and 1-359 truncation
mutants (Fig. 9B). Previously, it has been shown that single
mutations in either basic cluster of the bipartite NLS of nucleoplasmin
have little or no effect on nuclear transport, but mutations in both
basic clusters of the same molecule abolish nuclear transport (23). In
light of the fact that the truncations performed in this study indicate
that the TCPTP NLS is bipartite (Fig. 9B), we examined the
effect of incorporating the mutation of basic residues 350-352 and
deletion of basic residues 377-381 into the same molecule. We found
that when mutations in basic clusters I and II are combined, TCPTP is
evenly distributed between the nucleus and the cytoplasm (Fig.
9C), and its distribution is similar to that observed for
the 1-349 truncation mutant (Fig. 9B). These results are
consistent with the NLS of TCPTP being bipartite in nature and
reinforce the results obtained with the truncation mutants. Although
each basic cluster can function to a limited degree independently, both
basic clusters are required for optimal nuclear localization of TCPTP.
As such, the results demonstrate a direct correlation between the
binding of p97 and p116 and the bipartite nature of the TCPTP NLS.
Of the nuclear proteins in the data base, at least 50%
possess a bipartite NLS similar to that found in nucleoplasmin (22). The nucleoplasmin NLS comprises two basic clusters separated by a
10-residue spacer sequence. In the bipartite NLS of TCPTP, the two
basic clusters are separated by a spacer sequence of 24 residues. The
observation that this spacer in nucleoplasmin can be increased in
length to 22 residues without inhibition of nuclear import (23)
suggests that the motif in TCPTP satisfies the structural requirements
for a conventional bipartite NLS. However, the stable interaction we
observe between TCPTP and p97, together with the unusually long spacer
sequence, suggests the possibility that TCPTP may be unique and undergo
nuclear import by an alternative mechanism. Previous studies have shown
that p97 can function independently of importin- and import a
heterologous protein containing the IBB domain of importin-
(20,
21), raising the possibility that, in light of the stable association
of p97 with the TCPTP NLS, such an import mechanism may apply to the
phosphatase.
To test this possibility directly, import experiments were performed in
digitonin-permeabilized Madin-Darby bovine kidney cells (13, 14). In
these experiments, the ability of the purified nuclear import factors
to translocate recombinant TCPTP to the nucleus was assessed. It has
been shown that high concentrations of either p97 or importin- alone
may, to a variable extent, be sufficient to achieve import
(24).2 We found that p97
alone could not translocate TCPTP to the nucleus to a greater extent
than that achieved with importin-
alone. Optimal TCPTP import was
achieved only when both p97 and importin-
were present (Table
I). Similar results were obtained with
the SV40 T-Ag NLS (data not shown). Therefore, these results indicate, first, that the association of TCPTP with p97 does not simply mimic the
association of the importin-
IBB domain with p97 and, second, that
TCPTP can be imported by a process involving both p97 and importin-
,
at least in this in vitro assay system.
|
A knowledge of the intracellular distribution of nontransmembrane PTPs and the factors dictating their distribution may facilitate an understanding of the regulation and function of these enzymes. In the case of TCPTP, two distinct forms of the enzyme exist: a 48-kDa ER-associated form and a 45-kDa nuclear form. These TCPTP variants are generated by alternative splicing of the TCPTP transcript, and the two enzymes differ only at the extreme C terminus in the residues immediately following the TCPTP NLS. In the case of the 48-kDa TCPTP, the NLS is followed by a segment of 34 predominantly hydrophobic residues, whereas, in the case of the 45-kDa TCPTP, the NLS is followed by the 6-residue segment PRLTDT. We have isolated three proteins, p97, p116, and p120, that interact specifically with TCPTP at distinct as well as overlapping regions within the C-terminal segment.
Initially, the 20 hydrophobic C-terminal residues of 48-kDa TCPTP were identified as being responsible for the targeting of the protein to the particulate fraction of cell lysates (5, 6) and, more recently, have been shown to be necessary for directing the overexpressed protein to the ER (7). The fact that the 48-kDa form of TCPTP contains both the ER-targeting and NLS motifs but is found exclusively in the ER suggests that ER targeting is the dominant event. The C-terminal, hydrophobic segment of TCPTP, together with the second cluster of basic residues (377-381), is necessary for interaction with the binding protein p120. Interestingly, this interaction appeared specific in that the enzyme most closely related to TCPTP, PTP1B, which also contains a hydrophobic, C-terminal segment and localizes to the ER, did not associate with p120. The ability of p120 to interact with residues in the hydrophobic C terminus of TCPTP is consistent with a role in directing the enzyme to the ER. In addition, the fact that the binding site for p120 overlaps with the second cluster of basic residues in the bipartite NLS of TCPTP suggests that it may also play a regulatory role in controlling nuclear import by controlling the accessibility of the NLS to nuclear import factors. We are currently pursuing the identity of p120 to characterize further the function of its interaction with TCPTP.
The TCPTP-associated protein p97 was identified as the nuclear import
factor p97, otherwise known as importin- or karyopherin-
, which
is an essential factor in the nuclear transport of NLS-containing proteins (11, 14). Nuclear import is separated into two distinct steps.
The first is NLS-dependent binding to the nuclear pore complex at the nuclear membrane, and the second is translocation into
the nucleus. The binding step includes at least two cytoplasmic factors, the 54/56-kDa NLS receptor, otherwise known as importin-
or
karyopherin-
, and the nuclear import factor p97. Using the SV40 T-Ag
NLS as a model, numerous studies have reported that importin-
interacts directly with the NLS, whereas p97 interacts with
importin-
and docks the complex to the nuclear pore complex (16-18). The complex is believed subsequently to be translocated through the pore by an energy-dependent mechanism that
requires RanGTP (25-27).
In this study we report the binding of two distinct proteins, the
nuclear import factor p97 and p116, to the TCPTP NLS. This interaction
appears specific to the NLS of TCPTP in that in parallel binding assays
in vitro we did not detect interaction of p97 or p116 with
either the classical NLS of SV40 T-Ag or the standard bipartite NLS of
nucleoplasmin. The association of p97 with the TCPTP NLS was direct,
occurring independently of other proteins, and was also shown to occur
at physiological levels of expression. Both in precipitations from
[35S]Met-labeled cell lysates using GST-TCPTP constructs
as an affinity support and in direct binding assays in vitro
using purified GST-TCPTP NLS and importin-, we find that
importin-
binds poorly relative to the binding of p97. Furthermore,
TCPTP interacts with p97 at a site on the import factor that is
distinct from the importin-
binding site and therefore does not
simply mimic the association of p97 with the importin-
IBB domain.
Thus, it appears that the NLS of TCPTP participates in a unique, stable
interaction with p97. Moreover, we have provided evidence to indicate
that the TCPTP NLS is bipartite in nature, and we have demonstrated
that p97 and p116 interact with the two basic clusters lying at the termini of the NLS. The nuclear import factor p97 associated with residues 377-381 (basic cluster II), whereas p116 interacted with residues 350-352 (basic cluster I) and residues within basic cluster II. This illustrates a direct correlation between the binding of p97
and p116 to the basic clusters of the TCPTP NLS and NLS function.
However, the precise function of this interaction remains to be
ascertained. Our results suggest that there may be unique features to
the mechanism of nuclear import of TCPTP, in particular that p97 may
play a more direct role in the process than previously anticipated. In
this respect, it is interesting to note that p97 has been shown to
function independently of importin-
and to import a protein to which
has been fused the IBB domain of importin-
(20, 21). Nevertheless,
at least in the context of a nuclear import assay in permeabilized
Madin-Darby bovine kidney cells in vitro, we have
demonstrated that although p97 is essential for the import of TCPTP, it
is not sufficient. Optimal import required the presence of both p97 and
importin-
. However, it is important to bear in mind that these
assays in permeabilized cells, in which an excess of purified factors
is present, may not reflect accurately the normal physiological
condition and do not exclude the possibility of a more direct role for
p97 in the transport process in vivo.
TCPTP is one of few PTPs including PTP (28), PTP
/GLEPP-1 (29),
and DPTP61F (30) that exist in two forms with identical catalytic
domains but variable noncatalytic sequences that target the different
forms to distinct intracellular locations. The two TCPTP variants are
generated by alternative splicing and differ only at their extreme C
termini. In this study, we have isolated three proteins, p97, p116, and
p120, that interact with the TCPTP C terminus. p120 interacts with the
C-terminal residues that dictate an ER localization for the 48-kDa
TCPTP, whereas the nuclear import factor p97 and p116 interact with the
basic clusters of the TCPTP bipartite NLS, which targets the 45-kDa
TCPTP into the nucleus. Although p97 appears to be essential for
nuclear import of TCPTP, it is not sufficient. Thus, the identification
and characterization of p116 will be required to elucidate whether or
not the stable interaction of p97 and p116 with TCPTP reflects
a unique mechanistic aspect to the nuclear import of this
phosphatase.
We thank Drs. James Lorenzen and Edmond
Fischer for the TCPTP 45- and 48-kDa pSVL constructs, the TCPTP 45-kDa
C216S pUC18 construct, and the TCPTP 6228 antibody. We are grateful to
Dr. David Hill for the TCPTP CF4 antibody, to Drs. Harry Charbonneau and Luning Hao for the purified TCPTP 45-kDa protein and the pET-GST and pET-GST-TCPTP constructs, and to Dr. Colin Dingwall for the nucleoplasmin Bluescript construct and purified importin- protein. We thank Dr. Ryuji Kobayashi for the sequencing of p97 and p120 and
Drs. Michael Gutch and Anton Bennett for critical reading of the
manuscript.