(Received for publication, November 29, 1994; and in revised form, January 9, 1995)
From the
The mitogen-activated protein kinase (MAPK) also known as extracellular signal-regulated kinase (ERK) plays a crucial role in various signal transduction pathways. ERK is activated by its upstream activator, MEK, via threonine and tyrosine phosphorylation. ERK activity in the cell is tightly regulated by phosphorylation and dephosphorylation. Here we report the cloning and characterization of a novel dual specific phosphatase, HVH2, which may function in vivo as a MAP kinase phosphatase. The deduced amino acid sequence of HVH2 shows significant identity to the VH1-related dual specific phosphatase family. In addition, the N-terminal region of HVH2 also displays sequence identity to the cell cycle regulator, Cdc25 phosphatase. Recombinant HVH2 phosphatase exhibited a high substrate specificity toward activated ERK and dephosphorylated both threonine and tyrosine residues of activated ERK1 and ERK2. Immunofluorescence studies with an epitope-tagged HVH2 showed that the enzyme was localized in cell nucleus. Transfection of HVH2 into NIH3T3 cells inhibited the v-src and MEK-induced transcriptional activation of serum-responsive element containing promoter, consistent with the notion that HVH2 promotes the inactivation of MAP kinase. HVH2 mRNA showed an expression pattern distinct from CL100 (human homologue of mouse MKP1) and PAC1, two previously identified MAP kinase phosphatases. Our data suggest a possible role of HVH2 in MAP kinase regulation.
A group of protein serine/threonine kinases, known as
mitogen-activated protein kinase (MAPK) ()or extracellular
signal-regulated kinase (ERK), is acutely stimulated by various
extracellular signals, including mitogenic growth factors such as
insulin, epidermal growth factor (EGF), and phorbol esters (for review,
see (1, 2, 3, 4) ). ERK activation
is believed to play an essential role in mitogenic growth factor signal
transduction. Evidence indicates that ERK can phosphorylate nuclear
transcription factors (5, 6, 7) , protein
kinases(8) , cytoskeletal proteins(9) , and proteins
involved in regulation of cell growth(10, 11) ,
suggesting an essential role in cellular signal transduction. ERK must
be phosphorylated on both threonine and tyrosine residues to exert its
full enzymatic activity(12, 13) . A single protein
kinase, MEK, activates ERK2 by phosphorylating threonine 183 and
tyrosine 185(14, 15, 16) . Constitutive
activation of MEK can cause transformation in NIH3T3 cells and
differentiation in PC12 cells(17, 18) , demonstrating
the importance of the MAP kinase pathway in signal transduction. In
Swiss3T3 cells, MAP kinase reaches maximum activity approximately
5-10 min after EGF stimulation followed by a rapid
inactivation(19) . Western blotting demonstrated that the
amount of ERK protein did not change after mitogen stimulation,
suggesting that ERK is inactivated by post-translational
modifications(20) . Two protein phosphatases, MKP1/CL100 and
PAC1, have been implicated in dephosphorylation of ERK (21, 22, 23, 24, 25) .
Protein phosphatases are generally divided into Ser/Thr and Tyr phosphatases, based on the phosphoamino acid specificity. Unlike the protein kinases, protein Ser/Thr phosphatases share no sequence identity to the tyrosine-specific phosphatases. However, a growing number of phosphatases have recently been identified to dephosphorylate both Ser/Thr and Tyr residues (for review, see (26) ). The prototype of this dual specific phosphatase is the VH1 phosphatase encoded by the vaccinia virus(27) . Cellular proteins homologous to VH1 phosphatase have been identified. These include the cell cycle regulator Cdc25 (for review, see (28) ), the nitrogen-induced yeast YVH1(29) , and the human VHR(30) . KAP and Cdi1 were two dual specific phosphatases isolated by virtue of their interaction with the cyclin-dependent kinases(31, 32) . These enzymes have been implicated to play a role in cell cycle control.
An immediate-early gene, 3CH134, induced by serum and growth factors in mouse fibroblasts, was isolated and shown to have significant amino acid sequence identity to VH1(33) . The human homolog of 3CH134, CL100, was cloned by Keyse and Emslie (34) as an immediate-early gene in response to oxidative stress and heat shock. Another VH1-related immediate-early gene, PAC1, was isolated from mitogen activated T-cells (35) . CL100, 3CH134, and PAC1 have been demonstrated to specifically dephosphorylate threonine and tyrosine residues of ERKs(21, 22, 23, 24, 25) . A possible function of these mitogen-induced phosphatases may be to down-regulate ERK. Therefore, Sun et al.(23) have suggested the name of MKP1 (map kinase phosphatase) for 3CH134 as an indication of its biological function. Genetic studies in yeast Saccharomyces cerevisiae identified a dual specific phosphatase, MSG5, which inactivated the FUS3 and KSS1 kinases, two MAP kinase homologs in the yeast mating pathway(36) .
In this report, a novel dual specific phosphatase, HVH2 (for human VH1 homologous phosphatase 2), was isolated and characterized. The deduced HVH2 protein shares 62 and 55% sequence identity to CL100 and PAC1, respectively. Purified recombinant HVH2 specifically hydrolyzed the phosphothreonine and phosphotyrosine residues of the activated ERK1 and ERK2. HVH2 was found to be a nuclear protein and capable of blocking activation of a MAP kinase-regulated reporter gene expression.
Activated ERK1 and ERK2 were used as substrates for GST-HVH2. ERK (21.6 µg) was activated by GST-MEK2 (2.75 µg) in buffer B (18 mM HEPES, pH 7.5, 50 µM ATP, 10 mM magnesium acetate) at 30 °C for 20 min. The GST-MEK2 was depleted by absorption to glutathione-agarose (Sigma). Under these conditions, ERK was usually activated by more than 100-fold. The activated ERK was then used for HVH2 inactivation assay in 10 µl of buffer A at 30 °C for 10 min. Activity of HVH2-treated ERK was directly determined by the MBP kinase assay.
P-Labeled ERK1, ERK2, and GST-MEK2 were prepared by
autophosphorylation in the presence of
[
-
P]ATP in buffer B. Casein was
phosphorylated by either the catalytic subunit of protein kinase A or
P43
kinase as described(27) . ERK1 and ERK2
were also phosphorylated by GST-MEK2 in the presence of
[
-
P]ATP. Dephosphorylation of
P-labeled proteins was performed in 20 µl of buffer A
at 30 °C for 10 min using 3.8 microunits of GST-HVH2 or 1,000
microunits of PTP1. Samples were analyzed by SDS-PAGE and visualized by
autoradiography. Phosphoamino acid analysis was performed as described (42) .
The coding sequence of human ERK1 was subcloned into plasmid pALTER-1 (Promega) for site-directed mutagenesis. The catalytic essential lysine residue 71 of human ERK1 was mutated to arginine by oligonucleotide-directed mutagenesis (Promega) to produce a kinase-deficient ERK1*. The threonine 202 and tyrosine 204 were independently mutated to alanine and phenylalanine, respectively, in the kinase-deficient ERK1*. Mutations were confirmed by DNA sequencing and subcloned into pGEX-2T (43) for expression and purification. ERK1*, ERK1*T202A, and ERK1*Y204F were phosphorylated by GST-MEK2 as described above. Dephosphorylation of these mutant ERKs was performed as described for wild type ERK1. Dephosphorylation reactions were terminated by addition of SDS sample treatment buffer and resolved by SDS-PAGE. The samples were then transferred to nitrocellulose and quantitated by phosphoimaging or scintillation counting.
Activated ERK1 or ERK2 (0.4 µg) was inactivated by 5.9 microunits of GST-HVH2 in 30 µl of buffer A at 30 °C for 10 min. Sodium vanadate was added to inhibit HVH2. Half of the sample was directly used for MBP kinase assays. The other half was subjected to reactivation by 0.32 µg of GST-MEK2 in 20 µl of buffer B.
Figure 1: Sequence alignment of HVH2 with CL100 and PAC1. The deduced amino acid sequence of HVH2 was aligned with CL100 (34) and PAC1 (35) by the BESTFIT program of Wisconsin Genetics Computation Group. Conserved residues were highlighted. Gaps (as spaces) were introduced for the maximum alignment. The catalytically essential cysteine in all PTPs and VH1-related phosphatases is indicated by an asterisk (*).
The
deduced amino acid sequence of HVH2 displays 62 and 55% overall
sequence identity to the complete sequences of CL100 and PAC1,
respectively (Fig. 1)(34, 35) . The highest
sequence conservation occurs in the C-terminal half of the molecules,
where the catalytically essential cysteine (Cys for HVH2)
found in all protein tyrosine phosphatases is located. This C-terminal
domain also shares significant sequence identity to the active site
region of dual specific phosphatases such as VH1, YVH1, KAP, and Cdi1.
In contrast, the N-terminal 181 residues of HVH2 share only 33 and 25%
sequence identity to the corresponding regions of CL100 and PAC1,
respectively (Fig. 1). Interestingly, the N-terminal region of
HVH2 showed significant sequence identity with the cell cycle regulator
Cdc25 phosphatases. This sequence similarity has been observed in CL100
and MKP1(39, 51) . It is worth noting that the
catalytically essential cysteine in Cdc25 is absent in the N-terminal
regions of both CL100 and HVH2, suggesting that the catalytic domain of
HVH2 phosphatase resides in the C-terminal and not the N-terminal
region of the polypeptide.
Figure 2: A, inactivation of ERK1 and ERK2 by GST-HVH2. The activated ERK1 was inactivated by GST-HVH2 (closed circles) or PTP1 (open circles). The activated ERK2 was inactivated by GST-HVH2 (open triangles). ERK activity was determined by the MBP kinase assay. B, tyrosine and threonine phosphatase activity of HVH2. ERK1 was phosphorylated by GST-MEK2 before dephosphorylation (lane 1), or dephosphorylated by GST-HVH2 (3.8 µU, lane 2) or PTP1 (1 milliunit, lane 3). Positions of free phosphate, phosphoserine, phosphothreonine, phosphotyrosine, and origin are denoted by P, pS, pT, pY, and O, respectively.
If HVH2 dephosphorylated ERK on the same threonine and tyrosine residues which were recognized by MEK, the HVH2-dependent ERK inactivation should be reversible. To demonstrate this point, the HVH2-inactivated ERK2 was subjected to reactivation by MEK2 in the presence of 2 mM sodium vanadate, which inhibited the HVH2 activity. We observed that the HVH2-inactivated ERK2 could be quantitatively reactivated by MEK (not shown), indicating that HVH2 and MEK recognized the same residues on ERK.
An intriguing difference between the
activated ERK and other phosphoproteins was that the activated ERK
contained adjacent phosphothreonine and phosphotyrosine. It is possible
that the two neighboring phosphorylated residues serve as a recognition
determinant for HVH2. To test this hypothesis, ERK1 phosphorylated on
either threonine (ERK1*Y204F) or tyrosine alone (ERK1*T202A) was
utilized as a substrate for HVH2. Threonine 202 and tyrosine 204 in
ERK1 (53) correspond to threonine 183 and tyrosine 185 in ERK2
which are the activation-phosphorylation sites by
MEK(14, 15, 16) . ERK1*, a kinase-deficient
mutant, was phosphorylated on both threonine and tyrosine by MEK2 (Fig. 3B). ERK1*T202A, having threonine 202 substituted
by an alanine, was phosphorylated only on tyrosine while ERK1*Y204F,
having tyrosine 204 substituted by a phenylalanine, was phosphorylated
only on threonine (Fig. 3B). GST-HVH2 dephosphorylated
all three ERK1* mutants (Fig. 3A), suggesting that
double phosphorylations of adjacent threonine and tyrosine were not a
prerequisite for HVH2 recognition. However, HVH2 dephosphorylated ERK1*
and ERK1*T202A more efficiently than ERK1*Y204F (Fig. 3A), indicating that HVH2 preferred
phosphotyrosine over phosphothreonine. Interestingly, MEK also
phosphorylated tyrosine residues more efficiently than threonine
residues of ERK(54) . ()
Figure 3: A, dephosphorylation of ERK1* mutants. The MEK2-phosphorylated ERK1* mutants were dephosphorylated by HVH2 for various times (xaxis). Equal amounts (3,000 counts/min for each assay) of phosphorylated substrates were used for ERK1* (closed circles), ERK1*T202A (open triangles), and ERK1*Y204F (open circles). B, phosphoamino acid analysis of ERK1*, ERK1*T202A, and ERK1*Y204F. Kinase-deficient ERK1* mutants were phosphorylated by GST-MEK2 and subjected to phosphoamino acid analysis.
Figure 4: A, tissue distribution of CL100 mRNA. A multiple human tissue RNA blot was probed with CL100 cDNA for comparison to HVH2. Lanes: 1, heart; 2, brain; 3, placenta; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; and 8, pancreas. B, tissue distribution of HVH2 mRNA. The RNA blot is the same as stated in panel A and was probed with HVH2 cDNA. Lanes correspond to those in panel A. C, induction of HVH2 mRNA by phorbol 12-myristate 13-acetate but not by EGF. The arrow indicates the HVH2 mRNA (2.5 kb). Cyclophilin mRNA (the lower band) was detected as an internal control. Hep G2 cells were treated for 1 h by insulin-like growth factor (lane 1); EGF (lane 2); IBMX-forskolin (lane 3); phorbol 12-myristate 13-acetate (lane 4); hydrogen peroxide (lane 5); and control (lane 6). Hybridization signals were quantitated by phosphoimaging and normalized.
Figure 5: Nuclear localization of HVH2. Myc-tagged HVH2 was transfected into Hela cells and followed by immunofluorescence using a monoclonal anti-myc antibody. Panels are labeled as follows: A, immunofluorescence; B, nuclear staining by 4,6-diamidino-2-phenylindole; and C, phase contrast.
Figure 6: Inhibition of SRE dependent promoter activity by co-transfection of HVH2. A, v-src-induced transcriptional activation of a SRE-containing promoter was blocked by HVH2. The luciferase reporter plasmid was cotransfected with v-src and various amounts of pCMV-HVH2 myc. Luciferase activity was measured and normalized (yaxis). Results were representative of three independent experiments. B, inhibition of MEK-stimulated SRE promoter activity by HVH2. Cotransfection of HVH2 blocked MEK-induced luciferase expression. Luciferase activity in the absence (open bars) or presence (hatched bars) of 1 µg of pCMV-HVH2 myc. v-src (bars 1 and 2) and MEK (bars 3 and 4) used in the transfection are indicated.
Accumulating evidence supports that the mitogen-induced dual specific phosphatases play an important role in MAP kinase modulation (56) . The mouse MKP1 and human CL100 may be responsible for down-regulation of MAP kinase after growth factor stimulation(21, 22, 23, 25) , while the lymphocyte-specific PAC1 phosphatase may function in down-regulating MAP kinase in T-cells and B-cells(24, 35) . It is likely that different activators (MEKs) and inactivators (phosphatases) are required for the complex regulation of ERK. This is plausible for a number of reasons. First, ERK is known to be activated by numerous extracellular stimuli in a wide variety of cells. Second, ERK is a growing multi-enzyme family. One such example is the recently identified c-jun N-terminal kinase (JNK) which is related to ERK and activated by Thr/Tyr phosphorylation(57) . Different ERKs may be regulated by different activators (kinases) as well as inactivators (phosphatases). Furthermore, numerous different signal transduction pathways may use kinase cascades similar to the ERK pathway. For example, at least three distinct ERK-related signal transduction pathways have been identified in yeast, including the mating pheromone response, osmolarity regulation, and cell wall construction(58) . Existence of different MEKs and ERK-specific phosphatases provides a means by which the MAP kinase pathway could be differentially regulated.
In this report, we described the isolation of a novel dual specific phosphatase, HVH2, which showed significant sequence identity to CL100/MKP1 and PAC1 (Fig. 1). Several lines of evidence support that HVH2 is an ERK-specific phosphatase. First, HVH2 shows a high substrate selectivity toward MAP kinase and did not dephosphorylate any of the phosphoproteins tested except for the activated ERK1 and ERK2. The high substrate selectivity of HVH2 is analogous to CL100/MKP1 and PAC1 (21, 22, 23, 24, 25) . Second, HVH2 selectively dephosphorylated the threonine and tyrosine residues which were phosphorylated by MEK but not the autophosphorylated serine residue of ERK. Third, the nuclear localization is consistent with a role of HVH2 in MAP kinase regulation since the activated MAP kinase is known to be translocated into the nucleus(59) . Interestingly, PAC1 was also found in the cell nucleus (35) . Furthermore, ectopic expression of HVH2 blocked the activity of a SRE-containing promoter, consistent with the inactivation of MAP kinase. Caution should be taken to interpret the HVH2 overexpression data because nonspecific effects could occur. Nevertheless, the above observations strongly support that HVH2 phosphatase is likely to be an important component of the ERK/MEK signal transduction pathways, although the precise role of HVH2 in ERK inactivation remains to be elucidated.
PAC1 was maximally expressed in hematopoietic tissues and induced by T-cell activation(35) . MKP1/CL100 was induced by growth factors such as EGF, serum, and oxidative stress(22, 34, 39) . The MKP1/CL100 mRNA was highest in lung and also high in placenta and pancreas (Fig. 4A)(22, 39) . In contrast, HVH2 mRNA was induced by phorbol 12-myristate 13-acetate but not by EGF (Fig. 4B) and showed a tissue distribution pattern different from CL100. The HVH2 mRNA was highest in placenta followed by pancreas but was virtually undetectable in lung (Fig. 4B). A possible role of HVH2 in pancreas is consistent with the observation that cholecystokinin transiently activated MAP kinases in rat pancreatic acini(60) . Collectively, data from this report and previous studies demonstrate that different members of the ERK phosphatases, which are highly specific toward ERK, are expressed in different cells and tissues. The HVH2 described in this report represents one candidate of such phosphatases. These phosphatases are differentially regulated when cells exposed to wide variety of extracellular stimuli, providing additional mechanisms of MAP kinase modulation. Additional work on the regulation of these dual specific phosphatases will generate vital information to further elucidate signal transduction pathways utilizing MAP kinase.