(Received for publication, October 12, 1995)
From the
Dual specificity protein tyrosine phosphatases (dsPTPs) are a
subfamily of protein tyrosine phosphatases implicated in the regulation
of mitogen-activated protein kinase (MAPK). In addition to hydrolyzing
phosphotyrosine, dsPTPs can hydrolyze
phosphoserine/threonine-containing substrates and have been shown to
dephosphorylate activated MAPK. We have identified a novel dsPTP, rVH6,
from rat hippocampus. rVH6 contains the conserved dsPTP active site
sequence,
VXVHCXGX
RSX
AY(L/I)M,
and exhibits phosphatase activity against activated MAPK. In PC12
cells, rVH6 mRNA is induced during nerve growth factor-mediated
differentiation but not during insulin or epidermal growth factor
mitogenic stimulation. In MM14 muscle cells, rVH6 mRNA is highly
expressed in proliferating cells and declines rapidly during
differentiation. rVH6 expression correlates with the inability of
fibroblast growth factor to stimulate MAPK activity in proliferating
but not in differentiating MM14 cells. rVH6 protein localizes to the
cytoplasm and is the first dsPTP to be localized outside the nucleus.
This novel subcellular localization may expose rVH6 to potential
substrates that differ from nuclear dsPTPs substrates.
Extracellular signals, such as mitogenic growth factors, bind to
specific cell surface receptors that, in turn, initiate intracellular
signaling through activation of a series of protein kinases (reviewed
in (1) ). One such pathway involves the activation of
mitogen-activated protein kinase
(MAPK)()(
)(2) . MAPK is regulated by an
upstream kinase, MAPK/ERK kinase
(MEK)(
)(3, 4, 5) , which
phosphorylates MAPK on both Tyr and Thr residues within a TXY
phosphorylation motif (reviewed in (1) ). This
diphosphorylation activates MAPK and allows it to phosphorylate nuclear
transcription factors, protein kinases, cytoskeletal proteins, and
other cell growth-dependent substrates (see recent reviews in (1) and (6) ). Recently, a family of MAPKs, including
the MAPK-related proteins c-Jun N-terminal kinase/stress-activated
protein kinase and p38, has been identified and implicated in kinase
cascades that respond to mitogenic, differentiation, and stress-induced
signals (reviewed in (7) ).
MAPK and MAPK-related proteins can be inactivated by dephosphorylation of either Tyr or Thr residues (reviewed in (6) ). Two protein phosphatases, MAP kinase phosphatase (CL100) and PAC1, have been shown to dephosphorylate and inactivate MAPK both in vitro(8, 9, 10) and in vivo(10, 11) . These phosphatases belong to the protein tyrosine phosphatase (PTP) class of proteins, as defined by their conserved active site sequence, HCXAGXXR(S/T) (reviewed in (12) ). More specifically, MAPK phosphatase-1 and PAC1 are members of a subclass of PTPs commonly referred to as dual specificity protein tyrosine phosphatases (dsPTP) (reviewed in (13) ), which hydrolyze phosphate from Ser/Thr residues as well as from Tyr. Transcription of dsPTPs are induced by a variety of extracellular stimuli, where they may function in such processes as tissue regeneration(14) , serum and growth factor stimulation(9, 15, 16) , oxidative stress and heat shock response(15, 17) , nitrogen starvation(18) , and mitogen stimulation(19) . dsPTPs may also regulate cell cycle events, e.g. controlling entry into mitosis (20) and interacting with cyclin-dependent kinases (21, 22) . Dephosphorylation of MAPK and other MAPK-like proteins by dsPTPs suggests a function for dsPTPs in the regulation of cellular mitogenesis and differentiation.
In this study, we have identified a novel dsPTP from rat hippocampus, referred to as rVH6. rVH6 mRNA is detected in all tissues examined by Northern blot analysis. Recombinant rVH6 protein exhibits enzymatic activity against the artificial substrate, p-nitrophenyl phosphate (pNPP), and is able to dephosphorylate and inactivate MAPK in vitro. In COS-1 cells, expressed rVH6 protein localizes to the cytoplasm, and is thus the first dsPTP to be localized outside the nucleus. rVH6 mRNA is induced in PC12 cells following nerve growth factor (NGF)-mediated differentiation but is not induced following insulin or epidermal growth factor (EGF) mitogenic stimulation. The intermediate time course of rVH6 induction is distinct from other immediate-early gene dsPTPs and is closely correlated with the sustained activation and deactivation pattern of MAPK activity observed in NGF-mediated neuronal differentiation(23) . Interestingly, in proliferating MM14 myoblasts, rVH6 mRNA levels are expressed at high levels and then decline rapidly following commitment to muscle differentiation. Moreover, the loss of rVH6 message correlates with the ability of basic fibroblast growth factor (bFGF) to stimulate MAPK activity in MM14 muscle cells. The widespread expression of rVH6, as well as its induction by NGF and bFGF, delineates a potential role for rVH6 in regulating proliferation and differentiation.
GST-MEK2, MAPK (p44) and the kinase-deficient mutant, MAPK
K71R, were generous gifts from Dr. K. L. Guan. MAPK was activated by
incubation with GST-MEK2 as described previously(27) . The
ability of GST-rVH6 to inactivate MAPK was determined by incubation of
activated MAPK (0.2 µg) with increasing concentrations of GST-rVH6
in Buffer A (50 mM HEPES, pH 7.5, 0.1% 2-mercaptoethanol) for
10 min at 30 °C. The reactions were terminated by the addition of
sodium vanadate (final concentration, 2 mM), and the remaining
MAPK activity was determined as described(28) . Wild-type MAPK
and mutant MAPK K71R proteins were P-labeled by incubation
with GST-MEK2 in the presence of [
-
P]ATP
and purified as described(5) . The ability of GST-rVH6 to
dephosphorylate MAPK was determined by incubation of
P-labeled MAPK (0.2 µg) with increasing concentrations
of GST-rVH6 as described above. The reactions were stopped by the
addition of 5
Laemmli buffer, boiled, and resolved by
SDS-polyacrylamide gel electrophoresis. The proteins were transferred
to a polyvinylidene difluoride membrane (Schleicher & Schuell) and
exposed to film. After film development, radiolabeled MAPK was excised
from the membrane and subjected to phosphoamino acid
analysis(5) .
MM14 mouse muscle cells
were grown either in Ann Arbor from a cell stock generously provided by
Dr. Jeffrey Chamberlain or in Seattle from the original MM14 cell
stock. In both cases, the cells were grown in 0.6% gelatin-coated 140 -
20-mm plates containing Ham's F-10C nutrient medium supplemented
with 15% horse serum and 2 ng/ml bFGF (29) . For maximal cell
proliferation, MM14 cell medium was changed every 12 h, and the cells
were split before reaching 400,000 cells/plate. To initiate a
differentiation time course experiment, 550,000 cells were plated and
allowed to proliferate for 24 h. The medium was then removed, the
plates were washed with phosphate-buffered saline (PBS), and
differentiation medium (Ham's F-10C containing 2% horse serum, 1
µM insulin, without bFGF) was added. Differentiating cells
were fed every 24 h. For poly(A) RNA isolation,
20-30 plates were harvested per differentiation time point.
For detection of cellular MAPK activities, MM14 cells withdrawn from
serum and bFGF for the indicated lengths of time were stimulated
briefly (5 min) with either 10 ng/ml of bFGF or 1.5% horse serum in
F10C medium (control). The cells were harvested and lysed, and the high
speed supernatants (100,000 g) were fractionated over
DE-52 ``mini columns'' (Whatman) as described
previously(30) . MAPK activity was eluted from the columns with
0.2 M NaCl, normalized for protein, and assayed for activity
using myelin basic protein (Sigma) as substrate(30) .
Figure 1: A, nucleotide and predicted amino acid sequence of rVH6 cDNA. Nucleotide sequence is numbered on the left, beginning with the first nucleotide of the cDNA. Amino acids are numbered in italics on the left, beginning at the first predicted methionine. The in-frame stop codon is denoted by an asterisk. The PTP conserved active site is bold and underlined. Two putative nuclear export signals are underlined. B, alignment of rVH6 with other VH1-like PTPases. The amino acid sequence of rVH6 was aligned with CL100(17) , Pac-1(19) , VHR(35) , and VH1 (32) using the PILEUP program (Genetics Computing Group, Madison, WI). The black boxes denote conserved amino acid residues. The PTPase signature motif is double underlined, and the cdc25 homology domains (CH2 domains) are single underlined. Degenerate oligonucleotides used in PCR as described under ``Experimental Procedures'' are shown by arrows.
Preliminary Northern blot analysis (data not shown) using the rVH6 PCR fragment indicated that rVH6 was abundantly expressed in brain. Based on the high levels of expression in brain, a rat hippocampus cDNA library was screened with the rVH6 PCR fragment to isolate full-length cDNAs. Five hybridizing clones were isolated, four of which contained identical 2104-bp inserts and one of which contained a partial length insert of 1034 bp. The 2.1-kb cDNA sequence had a 1143-nucleotide (3) open reading frame (Fig. 1A) with a putative initiator methionine in a region that matched the Kozak sequence motif (nucleotides 355-363; Fig. 1A)(33) . No other methionines were observed 5` to this methionine in any other reading frame. In addition, 5` to the Kozak sequence and 3` to the in-frame stop codon (nucleotides 1504-1506; Fig. 1A), stop codons were observed in all three reading frames. No polyadenylation signal or poly(A) tail was observed in any rVH6 cDNA clone isolated. Based on the 3.2-kb message size observed in Northern blot analysis (Fig. 2), up to 1.1 kb of 5` and 3` untranslated sequence was not present in the isolated cDNA clones. Northern blot analysis using the rVH6 riboprobe revealed that the mRNA is expressed as a 3.2-kb transcript in all the tissues examined (Fig. 2). Relatively higher levels of expression were seen in brain and spleen, with lower levels in heart, lung, liver, and skeletal muscle. Very low levels of rVH6 mRNA were also detected in kidney and testis. A similar size rVH6 RNA transcript was also observed in a mouse multiple-tissue Northern blot (data not shown). The rVH6 mRNA tissue distribution between rat and mouse was similar, with the exception that mouse kidney and skeletal muscle expressed higher levels of rVH6 (data not shown).
Figure 2:
Tissue distribution of rVH6. Rat multiple
tissue Northern blot (Clontech) of poly(A) RNA (2
µg/lane) was screened with a rVH6 antisense probe as described
under ``Experimental Procedures.'' RNA size markers are shown
to the left.
Data base searches with the deduced amino acid sequence of rVH6 (Fig. 1B) indicate that rVH6 is similar to all known
dsPTPs. The rVH6 cDNA predicted open reading frame codes for a protein
of 381 amino acids (42,315 Daltons), which is similar in size and
amino acid identity to CL100 (36%,(17) ), Pac-1
(35%,(19) ), hVH2 (35%, (27) ), and hVH3
(33%,(16) ). rVH6 contains the active site sequence,
VXVHCXXGXXRSXXXXXAY(L/I)M (Fig. 1B), characteristic of virtually all
dsPTPs(34) . The structure of rVH6 can be divided based on its
amino acid alignment with other dsPTPs (Fig. 1B) into a
181-amino acid COOH-terminal catalytic domain and a 200-residue
NH
-terminal extension. The rVH6 catalytic domain possesses
amino acid identities to hVH2 (45%), CL100 (44%), Pac-1 (43%) hVH5
(42%,(34) ), hVH3 (38%) VHR (35%,(35) ), and VH1 (27%).
The NH terminus of rVH6 contains two regions of amino
acid similarity to the cell cycle regulator phosphatase,
cdc25(36, 37) . These two regions (underlined in Fig. 1B) are referred to as cdc25 homology
domains 2 (CH2 domains) (37) and have been observed in the
NH
terminus of several other dsPTPs (CL100, Pac-1, hVH2,
hVH3, and hVH5). The function of these CH2 domains is unknown.
Interposed between the NH
-terminal CH2-containing, and
COOH-terminal catalytic domains, is a serine-rich (30%) amino acid
region (amino acids 150-210; Fig. 1B) that also
contains two putative proline-directed serine kinase substrate
recognition motifs, XSP, (amino acids 162-164 and
200-202; Fig. 1B) (reviewed in (1) ). In
addition, the NH
-terminal domain of rVH6 contains two
putative nuclear export motifs, LXLXLXXL and
LXXLXLXXL (Fig. 1A),
implicated in the function of proteins that export specific proteins
and RNAs from the nucleus(38, 39) . No other
significant amino acid similarity to other Genbank protein sequences
was observed in the NH
-terminal domain of rVH6.
Figure 3: Kinetic analysis of rVH6 against pNPP. A, hydrolysis of pNPP with increasing concentrations of recombinant GST-rVH6 (closed circles) or GST (open circles). B, hydrolysis of pNPP by GST-rVH6 (30 µg) in the absence (closed circles) or presence (open circles) of 1 mM sodium vanadate. Enzyme assays and determination of rVH6 kinetic properties were as described under ``Experimental Procedures.'' Data points are the means of triplicate experiments with S.E. < 5%.
Figure 4:
Inactivation of MAP kinase by rVH6. A, increasing amounts of purified recombinant GST-rVH6 were
assayed for their ability to inactivate p44 MAP kinase (0.2 µg) in
the absence (closed circles) or presence (open
circles) of 1 mM sodium vanadate. The control MAPK
activity was measured without GST-rVH6. B, autoradiograph of a
SDS-polyacrylamide gel showing the dephosphorylation of P-labeled wild-type (WT) and mutant K71R MAP
kinase treated with mutant C293S GST-rVH6, wild-type GST-rVH6 + 1
mM sodium vanadate, and increasing concentrations of GST-rVH6. C, phosphoamino acid analysis of
P-labeled MAP
kinase treated with indicated amounts of GST-rVH6. Data points in A are the mean values of triplicate experiments with S.E. <
5%.
To
determine the specific amino acids dephosphorylated by rVH6, P-labeled MAPK previously incubated with GST-rVH6 was
subjected to phosphoamino acid analysis (Fig. 4C). The
removal of label from tyrosine residues closely correlated with the
dephosphorylation and inactivation of MAPK. Surprisingly, very little
of the phosphothreonine residues were dephosphorylated by rVH6 in 10
min. However, dephosphorylation of phosphothreonine residues could be
observed with rVH6 treatment greater than 1 h (data not shown). This
rapid tyrosine dephosphorylation and slow threonine dephosphorylation
pattern closely resembles the dephosphorylation of pTyr and pThr on
p34
by the dsPTP, cdc25(20) . Under similar
conditions, CL100, hVH2, and hVH3 were able to rapidly dephosphorylate
both tyrosine and threonine on activated
P-labeled MAPK (8, 16, 27) .
Figure 5:
Induction of rVH6 expression in
NGF-mediated neuronal differentiation. Autoradiograph (A) and
quantitation (B) of a PC12 cell poly(A) RNA
(2 µg/lane) Northern blot hybridized with rVH6 and cyclophilin
riboprobes. PC12 cells were serum-starved for 18 h and induced to
differentiate with NGF (100 ng/ml) treatment for the indicated times.
rVH6 message levels were quantitated and normalized against endogenous
cyclophilin mRNA as described under ``Experimental
Procedures.'' Fold stimulation of rVH6 mRNA levels was calculated
relative to levels at time 0 in the absence of NGF. The data shown are
representative of a typical experiment repeated three
times.
Figure 6:
rVH6
expression in MM14 muscle cell differentiation. Autoradiograph (A) and quantitation (B) of a MM14 cell
poly(A) RNA (1 µg/lane) Northern blot hybridized
with rVH6 (hashed bars), muscle creatine kinase (black
bars), and cyclophilin riboprobes. Proliferating cells (0 h) were
withdrawn from bFGF and high concentrations of serum. Isolation and
analysis of the RNA at the indicated time points was performed as
described under ``Experimental Procedures.'' Lane +F was treated for 48 h in the presence of 2 ng/ml bFGF and 2% serum,
whereas lane +S was treated with 15% serum and no bFGF.
RNA levels were normalized against endogenous cyclophilin mRNA. The
data shown are representative of a typical experiment repeated twice
with similar results.
Figure 7: MAP kinase activity during MM14 muscle cell differentiation. MM14 cells were withdrawn from bFGF and high serum and harvested at the indicated times. Following a 5-min stimulation by bFGF (10 ng/ml), cell extracts were prepared, and MAPK activity was isolated and measured as described under ``Experimental Procedures.'' The data are presented as fold stimulation of MAPK activity (bFGF-stimulated MAPK activity divided by non-bFGF-stimulated MAPK activity). Similar results were seen in two separate experiments.
Figure 8:
rVH6 localizes to the cell cytoplasm. The
localization of expressed epitope-tagged rVH6 or hVH3 in COS-1 cells
was visualized by immunofluorescence. HA-tagged rVH6 (A),
anti-HA antibody control (B), c-myc-tagged hVH3 (C), and anti-myc antibody control (D) were
detected with a fluorescein-conjugated anti-mouse secondary antibody as
described under ``Experimental Procedures.'' COS-1 cell
immunofluorescence was photographed under a 40 microscope
objective.
Tissue regeneration and serum stimulation have been shown to
induce the expression of
dsPTPs(9, 14, 15, 16) . Certain
dsPTPs also appear to play a role in proliferation and differentiation
by regulating the activities of MAPKs (reviewed in (13) ). In
olfactory epithelium, olfactory neurons undergo continuous
regeneration, proliferation, and differentiation to replace damaged
olfactory receptors(47) . We therefore considered rat olfactory
tissue as a likely source of novel dsPTPs. rVH6 was identified from rat
olfactory cDNA by degenerate oligonucleotide PCR and subsequently
cloned from rat brain cDNA. The cDNA codes for a protein that shares
amino acid identity with other dsPTPs, including the extended PTP
active site consensus sequence,
VXVHCXXGXXRSXXXXXAY(L/I)M (34) . All of the amino acids previously shown to be important
for dsPTP catalytic activity are conserved in rVH6, including
Cys, the catalytic cysteine that functions as the active
site nucleophile, and Ser
and Asp
, both
involved in hydrolysis of the thiol-phosphate
intermediate(48, 49) . Similar to other dsPTPs, rVH6
contains two CH2 domains in an NH
-terminal extension.
Although the functions of the CH2 domains are unknown, it has been
suggested that they may confer substrate specificity or function via
interactions with other regulatory proteins(36) . The rVH6
protein also contains a serine-rich region between the
NH
-terminal CH2 and COOH-terminal catalytic domains. This
amino acid region containing two putative proline-directed serine
kinase substrate recognition motifs may be amenable to phosphorylation,
thereby potentially contributing to the regulation of the protein.
Bacterially expressed GST-rVH6 protein possesses PTP activity and
hydrolyzes the artificial substrate, pNPP. The affinity of rVH6 for
pNPP is 10- and 2-fold lower than that of VHR and cdc25, respectively (40, 41) . The k for pNPP
hydrolysis is also 80-fold slower than VHR and more closely resembles
cdc25 with only a 1.4-fold lower turnover rate. The differences in
kinetic parameters between rVH6 and VHR may demonstrate a higher
selectivity of rVH6 for its endogenous substrate. In contrast, rVH6
appears to have activity similar to CL100 in dephosphorylating and
inactivating MAPK in vitro. CL100 inactivates MAPK both in
vitro and in vivo and has therefore been suggested to be
the physiological MAPK phosphatase-1(8, 11) . rVH6
inactivates MAPK at similar protein concentrations as CL100, 10 nMversus 7 nM, respectively(42) ,
suggesting that rVH6 may also be able to physiologically recognize MAPK
as a substrate and regulate its activity in vivo.
Several
differences between rVH6 and other dsPTPs are noted. Although rVH6 will
rapidly dephosphorylate phosphotyrosine in P-labeled MAPK,
the rate of hydrolysis for phosphothreonine is much slower than with
other dsPTPs(8, 16, 27, 40) and
more closely resembles the dephosphorylation of p34
by
the dsPTP, cdc25(41) . Although dephosphorylation of
phosphotyrosine is sufficient by itself to inactivate MAPK, the
differences in rate of phosphothreonine hydrolysis may reflect
differences in substrate specificity.
The intracellular localization
of rVH6 is novel. Previously, expressed epitope-tagged dsPTPs (Pac-1,
MAPK phosphatase-1, hVH2, and hVH3) have been immunolocalized to the
nucleus(16, 19, 27, 50) . rVH6 is
the first dsPTP to be localized outside the nucleus. Interestingly,
rVH6 contains two putative nuclear export signals (38, 39) (Fig. 1A), one of which falls
within the serine-rich amino acid extension found between the
NH-terminal CH2-containing domain and the COOH-terminal
catalytic domain of rVH6 (Fig. 1B). We were unable to
find these motifs in the NH
-terminal regions of any other
mammalian dsPTP, suggesting that these nuclear export signals may be
responsible for the novel cytoplasmic localization of rVH6. This
intracellular localization could enable rVH6 to interact with potential
substrates different from the nuclear dsPTPs, including the
inactivation of MAPK prior to its nuclear translocation. In addition,
30-50% of MAPK activity has been shown to be associated with the
microtubule cytoskeleton(51) . This cytoskeletal MAPK could,
therefore, be a potential substrate for a cytoplasmic dsPTP. Recently,
other diphosphorylated MAPK family members have been
identified(52, 53, 54) . One of these
members, p38, has been expressed and immunolocalized to both the
cytoplasm and nucleus in COS-1 cells(55) . Interestingly,
following environmental stress (ultraviolet radiation), p38 shows an
increased localization to the perinuclear region of the cytoplasm, an
area rich in expressed rVH6. As more potential substrates are
identified and examined, the question of rVH6 substrate specificity may
be addressed.
Based on the abundant expression of rVH6 mRNA in brain, we wished to determine whether rVH6 was also expressed in PC12 cells and whether it played a potential role in cell proliferation or differentiation. MAPK can be activated in PC12 cells by both mitogenic (EGF or insulin) or differentiation (NGF) stimuli. The duration of MAPK activation has been suggested as the controlling factor between proliferation (transient activation) or differentiation (sustained activation)(23) . Some evidence suggests that the Ser/Thr phosphatase, PP2A, is involved in the rapid deactivation of MAPK activity in PC12 cells(43) . dsPTPs, CL100, and hVH5 are transcriptionally induced as immediate early genes in response to EGF, insulin, or NGF(34, 42, 43) . rVH6, however, is not expressed under mitogenic conditions following EGF or insulin stimulation of serum-starved PC12 cells. Rather, stimulation to differentiate with NGF results in sustained rVH6 mRNA expression 1-2 h later than the dsPTP immediate early genes. Moreover, the time course of rVH6 mRNA expression parallels the sustained activation and delayed inactivation of MAPK observed in NGF-treated PC12 cells (23) . The inability of EGF or insulin to induce rVH6 expression, as well as the time course of rVH6 expression induced by NGF, suggests that rVH6 may play a role in PC12 neuronal differentiation.
In the mouse skeletal muscle cell line, MM14, FGF represses muscle differentiation and together with serum maintains cell proliferation (45) . It has recently been shown that bFGF activates MEK but not MAPK in MM14 myoblasts that have been withdrawn from mitogens for 3 h(46) . However, both MEK and MAPK are activated by bFGF after 10 h of mitogen withdrawal. The inability of bFGF to activate MAPK in myoblasts appears to be due to a PTP activity that decreases with time following mitogen withdrawal(46) . Interestingly, rVH6 mRNA is highly expressed in proliferating myoblasts but rapidly declines following mitogen withdrawal. After 6 h in differentiation medium, a time at which bFGF is able to stimulate MAPK activity (Fig. 7), the levels of rVH6 message have dropped by 80% (Fig. 6). Thus, expression of rVH6 is inversely correlated with the ability of bFGF to stimulate MAPK activity in MM14 cells. In contrast to PC12 cells, rVH6 expression in MM14 cells is highest in proliferating cells and lowest in differentiated cells.
In this study, we have identified a novel dual specificity phosphatase that is expressed in many tissues and appears to have a unique intracellular localization. rVH6 dephosphorylates and inactivates MAPK in vitro. The novel cytoplasmic localization of rVH6 may allow it to interact with substrates that are distinct from the substrates of nuclear dsPTP. Our results also suggest that rVH6 may play a role in promoting proliferation and repressing differentiation in MM14 muscle cells while promoting differentiation in PC12 neuronal-like cells.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U42627[GenBank].