©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Multiple Dual Specificity Protein Tyrosine Phosphatases Are Expressed and Regulated Differentially in Liver Cell Lines (*)

(Received for publication, September 8, 1994; and in revised form, November 11, 1994)

Seung P. Kwak Jack E. Dixon (§)

From the Department of Biological Chemistry, The University of Michigan, Ann Arbor, Michigan 48109-0606

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

An emerging subclass of protein-tyrosine phosphatases (PTPases) exhibits sequence identity to the vaccinia H-1 (VH-1) gene product. These VH-1-like PTPases possess the canonical HCXAGXXR(S/T) sequence common to all PTPases, but unlike other PTPases they exhibit dual catalytic activity toward phosphotyrosine and nearby phosphothreonine residues in substrate proteins. We have isolated a novel VH-1-like PTPase, hVH-3, from the human placenta and compared various aspects of its expression with previously isolated members of this subfamily. The mammalian members of this subfamily including hVH-3 commonly localize to the nucleus and exhibit catalytic activity toward phosphorylated extracellular signal-regulated kinase. However, while the expression of some VH-1-like PTPases is extremely transient and independent of protein synthesis, hVH-3 expression is sustained over 3 h after being cell stimulated. Tissue-specific expression of hVH-3 is also distinct from other VH-1-like PTPases. Although VH-1-like PTPases have overlapping substrate specificity, there are differences in their mRNA regulation, response to extracellular stimuli, and tissue-specific expression, suggesting they serve specific roles in cellular function.


INTRODUCTION

Extracellular signal-regulated kinase (ERK) (^1)plays a key role in mediating signal transduction of a variety of extracellular stimuli. Since this kinase requires dual phosphorylation of a tyrosine and a neighboring threonine residue for activation(1) , phosphatases with specificity for phosphorylated residues that are in close apposition may be critical for regulation of ERK. The vaccinia H-1 gene product (VH-1) is one such enzyme. VH-1 contains the peptide motif HCXAGXXR common to all protein tyrosine phosphatases (PTPases) but differs from other PTPases in its ability to hydrolyze substrates phosphorylated on both Tyr and Ser/Thr residues(2) . Recent isolation of phosphatases that exhibit high sequence identity to VH-1 indicates that the VH-1-like PTPases represent a novel subfamily that perhaps functions to regulate the activity of signal transduction proteins like ERK.

VH-1-like PTPases have been isolated from a diverse group of organisms including yeast (yVH-1 and MSG-5)(3, 4) , pox viruses(5) , and mammals (VHR(6) , Pac-1(7) , and CL100(8, 9) ). The mammalian VH-1-like PTPases exhibit similar features. For instance, CL100 (also termed MKP1 or ERP (10, 11) ) and Pac-1 are expressed during mitotic stimulation. The time course of their mRNA expression is characteristic of immediate early genes, which do not require protein synthesis for induction and possess a short mRNA half-life(7, 12) . Both PTPases exhibit activity toward phosphorylated ERK-1(10, 13, 14) . More recently, VHR has also been shown to dephosphorylate ERK-1 in vitro(15) . Finally, CL100, Pac-1, and VHR are all localized in the nucleus (7) . (^2)These observations suggest that the mammalian VH-1-like PTPases may have overlapping substrate specificities.

Recent evidence suggests that multiple ERK-like kinases are expressed in the same tissues but serve specific roles in cellular function. In yeast for instance, three ERK homologues are expressed that appear to function in three distinct signaling pathways involved in regulation of mating, osmosis, and cell wall formation(16, 17) . Multiple VH-1-like PTPases are also present in yeast and mammalian cells, leading one to suspect that these PTPases, despite exhibiting many similar features, are differentially regulated and function in parallel with ERK-like kinases in their specified pathways.

In the present study, we have characterized a novel dual specificity PTPase, hVH-3 (human VH-1 like clone 3), that is co-expressed with another VH-1-like PTPase, VHR, in liver cell lines. We compared the properties of hVH-3 and other VH-1-like PTPases in a variety of cells and in all cases observed similar cellular localization and substrate specificity. However, differences were found among the VH-1-like PTPases in tissue distribution and mRNA regulation in response to extracellular stimuli. These observations indicate that although some features are common to VH-1-like PTPases, differences exist that may distinguish their functional roles in vivo.


MATERIALS AND METHODS

cDNA Library Screening

A 500-base pair DNA fragment encoding the active site of clone hVH-3 (chromosome assignment 10q25) (18) was labeled with random primers in the presence of [P]dCTP to be used as a probe to screen a human placental library (Stratagene). Approximately 1 million plaques were lifted on nitrocellulose filters (Schleicher & Schuell) and screened as previously described(19) . Filters were washed in a final condition of 60 °C for 1 h and exposed on film (Kodak X-AR); the positive plaques were purified and sequenced using Sequenase V2.0 (Stratagene). Alignments between the novel PTPase protein and various VH-1-like phosphatases were done using the PILEUP algorithm (GCG version 7.0) and modified by visual inspection.

Bacterial Expression of Fusion Protein (GST-hVH-3-His)

The hVH-3 cDNA was subcloned into pGEX-EX, a plasmid modified from pGEX-KT by insertion of four additional cloning sites at the polylinker, to produce a fusion protein containing glutathione S-transferase at the amino terminus. A polyhistidine tag was added to the carboxyl terminus of hVH-3 by polymerase chain reaction to add a second means of affinity purification. A pair of oligonucleotides (5`-AAATCTAGATTAATGATGATGATGATGATGGCAGGATGTGGCCGTTGC-3` and 5`-TGCTGGCACCGGTGCCTAC-3`) were used to amplify a 100-base pair fragment of hVH-3 encoding 6 additional histidine residues at the carboxyl end. The final fusion protein, GST-hVH-3-His, was produced in Escherichia coli strain BL21 as previously described(20) . Purification was performed initially by sequential passage through glutathione-agarose and nickel-agarose columns. Subsequently, we found it sufficient to purify the protein over the nickel-agarose column as described by the manufacturer (Qiagen, Chatsworth, CA).

Enzyme Activity Assay

Hydrolysis of para-nitrophenol phosphate by hVH-3 was performed as described elsewhere(21) . Protein substrate ERK1, an ERK1 mutant devoid of intrinsic kinase activity, was a generous gift from Dr. K. L. Guan. ERK1 was radioactively labeled by recombinant MEK2 and purified as described(14) . P-Labeled ERK1 (2 µg) was incubated with partially purified hVH-3 in buffer containing sodium succinate (200 mM, pH 6.5), EDTA (1 mM) for 5 min at 30 °C. The reaction was stopped by addition of 5 times Laemmli buffer and resolved by SDS-polyacrylamide gel electrophoresis. Phosphoamino acid analysis of radiolabeled ERK1 was performed as previously described (14) .

Cellular Localization of hVH-3

A construct was made in plasmid pCMVneo containing hVH-3 cDNA with carboxyl-terminal, c-Myc epitope tag(22) . A monoclonal antibody (9E10) that specifically recognizes the epitope sequence EQKLISEEDL was purified from cultured hybridoma cells. Plasmid DNA (5 µg) was transfected into human cervical carcinoma (Hela) or monkey kidney epithelial (Cos-1) cells using 6 µl of Lipofectin according to manufacturer's instructions (Life Technologies, Inc.). After 6 h of transfection, cells were washed in Dulbecco's phosphate-buffered saline (PBS), then allowed to recover in Dulbecco's modified Eagle's medium with 10% calf serum. 2 days following transfection, cells were washed once with PBS and fixed in 3% formaldehyde for 10 min, followed by methanol:acetone treatment (1:1) for 5 min. The cells were washed 3 times in PBS, incubated in PBS containing bovine serum albumin (1%) and saponin (0.1%) with monoclonal Ab 9E10 (10 ng/ml) for 1 h. The secondary horse anti-mouse antibody conjugated to Texas Red (Vectastain, Burlingame, CA) was used at 1:200 dilution.

Hepatoma Cell Cultures

HepG-2 cells (ATCC) were grown in minimum essential-alpha medium supplemented with 10% fetal calf serum (Life Technologies, Inc.). Reuber H4 cells (ATCC) were grown in minimum essential medium supplemented with 10% fetal calf serum. Cells were serum-deprived for 24 h prior to experimentation and either harvested (unstimulated) or treated for 1 h with epidermal growth factor (EGF) (1 µg/ml), insulin-like growth factor-1 (163 ng/ml), insulin (100 nM), glucagon (100 nM), interleukin-6 (100 ng/ml), phorbol 12-myristate 13-acetate (500 nM), hydrogen peroxide (200 µM), or forskolin/isobutyl-methylxanthine (25 µM/500 µM).

Northern Blot Analysis

A human multiple tissue Northern blot was commercially obtained (Clontech). Northern blots of cultured hepatoma cells were prepared by extraction of total RNA in TRIzol solution (Life Technologies, Inc.), followed by purification of poly-adenylated mRNA with Poly(A)Tract kit (Promega) and electrophoresis in formaldehyde-agarose gel as previously described (23) . Blots were hybridized overnight at 45 °C in 50% formamide, 4 times SSC, 5 times Denhardt's solution, 10 µg/ml sheared salmon sperm DNA. A fragment of hVH-3 cDNA spanning nucleotides 1-704 was used as probe. The membranes were washed in a final condition of 0.1 times SSC, 0.1% SDS at 60 °C and exposed on film for 2-5 days at -70 °C with intensifying screens. The RNA bands were quantitated by optical densitometry (NIH Image, v1.53).


RESULTS AND DISCUSSION

Cloning of a Novel PTPase That Exhibits Homology to CL100 and Pac-1

We have previously reported the partial characterization of novel human PTPase genes and their chromosomal localization(18) . We have subsequently characterized the cDNA of one gene (chromosome assignment 10q25) in detail. Sequences from three overlapping clones were combined to generate a final cDNA of 2.1 kilobases encoding hVH-3, a novel human VH-1-like PTPase. The predicted hVH-3 protein (384 amino acid residues) exhibited the highest degree of sequence similarity to the mammalian members of the VH-1 subfamily of PTPases. hVH-3, like mammalian VH-1-like PTPases CL100 and Pac-1, contained the PTPase signature motif near the carboxyl end of the protein (Fig. 1A, regionC). Additional regions of sequence similarity were found among hVH-3, CL100, and Pac-1. In particular, the conserved sequences at the amino terminus (Fig. 1A, regions1 and 2) are noteworthy as they also align with regions of cdc25, a phosphatase involved in cell cycle regulation(19, 24) . While little is known regarding the functional significance of these sequences, their presence at the amino terminus distinguishes hVH-3, CL100, and Pac-1 structurally from VHR and other members of the VH-1 subfamily. Other regions of hVH-3, including the AYLM motif, were conserved among all members of the VH-1 subfamily (Fig. 1A, regions3 and 4). The importance of these residues is yet to be elucidated.


Figure 1: A, alignment of hVH-3 with other VH-1-like PTPases. Blackboxes denote residues conserved among the three closely related members, hVH-3, CL100 (human), and Pac-1 (human), or among all members including VHR (human), VH-1 (vaccinia H-1 product), and yVH-1 (yeast). The region containing the PTPase signature motif is bracketed (C). Bracketedregions1-4 and asterisks are discussed under ``Results.'' B, PTP1B sequence near the PTPase motif (bold). The P-loop is formed between a beta sheet (arrow) and an alpha helix (openbox) and followed by 2 conserved residues.



The presence of the canonical PTPase motif (HCXAGXXR(S/T)) suggests that VH-1-like phosphatases and tyrosine-specific phosphatases like PTP1B and Yersinia PTPase YOP51 have similar catalytic mechanisms and may share structural identity near this region. Structural elucidation of PTP1B (25) and Yop51 (26) has provided evidence that active site motif HCSAGIGRS is situated between a beta sheet and an alpha helix and forms a loop structure (Fig. 1B). This ``P-loop'' is essential for recognition of the phosphate moiety on the substrate. Residues within the P-loop of PTP1B that interact directly with the phosphate group, including the catalytic cysteine (Cys) and a nearby arginine (Arg), and those that orient the phosphate binding loop via hydrogen bond formation (His, Ala, and Ser) are conserved in VH-1-like PTPases. Corresponding residues perform similar functions in the Yersinia PTPase structure(26) . Other important residues of PTP1B include Arg and a pair of closely situated Gln residues (Gln and Gln) (Fig. 1B). These residues are conserved in Yop51 and other tyrosine-specific phosphatases and function to facilitate catalysis (25, 26) . Mammalian VH-1-like PTPases also possess a conserved Arg residue and a pair of Gln residues at approximately similar distance from the catalytic site (Fig. 1, asterisks). The presence of these key residues and the motif in the VH-1-like PTPases leads to the hypothesis that this subclass of PTPase also forms an active site loop configuration and suggests that the conserved Arg residue and the pair of Gln residues found at the carboxyl terminus of VH-1 subfamily serve similar functions as they do in tyrosine-specific phosphatases.

Catalytic Properties and Cellular Localization of hVH-3 Are Similar to Other VH-1 PTPases

Since hVH-3 shared several amino acid motifs common to other PTPases, it was not surprising to find that for the recombinant protein hVH-3, the optimal pH for catalysis of para-nitrophenol phosphate was acidic as well (pH 5.2, data not shown). Furthermore, hVH-3 blocked the kinase activity of wild-type ERK1 that was induced by MEK2 (data not shown). Consistent with this observation, hVH-3, like CL100 and Pac-1, was capable of dephosphorylating MEK-activated ERK1 in a concentration-dependent manner (Fig. 2A). Co-incubation with Na(3)VO(4) effectively blocked dephosphorylation of ERK1 by hVH-3, while NaF and NaPPi were not very effective. Phosphoamino acid analysis confirmed that ERK was phosphorylated by MEK2 at both Thr and Tyr residues, with phosphorylation occurring preferentially at the Tyr residue (Fig. 2B). PTPase hVH-3 dephosphorylated ERK1 at both residues, confirming its activity as a dual specificity PTPase.


Figure 2: A, enzyme activity of bacterially expressed hVH-3. Dephosphorylation of kinase-deficient ERK1 (2 µg) by increasing amounts of recombinant hVH-3 (lanes1-3; 0, 50, 100 ng) is shown. Dephosphorylation of ERK1 (1 µg) by hVH-3 can be blocked by Na(3)VO(4) (lane4, ERK alone; lane5, hVH-3 (20 ng) + NaF (100 mM), NaPPi (2 mM); lane6, hVH-3 (20 ng) + Na(3)VO(4) (1.5 mM). B, phosphoamino acid analysis of P-Labeled ERK shown above. ERK1 was phosphorylated by recombinant MEK2 at Tyr and Thr residues; hVH-3 dephosphorylated ERK1 at both residues.



We subsequently determined the subcellular localization of hVH-3 by transiently transfecting an epitope-tagged hVH-3 cDNA into Cos-1 and Hela cells. Immunofluorescence histochemistry using an antibody raised against the c-myc epitope revealed that hVH-3 is found primarily in the nucleus (Fig. 3A). Transfected CL100 produced a similar localization pattern, suggesting that the function of this PTPase subfamily is restricted to the nucleus (Fig. 3B). Fluorescence was undetectable from untransfected cells or cells transfected with the vector alone (data not shown).


Figure 3: Indirect immunofluorescence analysis of transfected hVH-3 expression in Hela cells. Expression of epitope-tagged hVH-3 (panelA) and CL100 (panelB) was detected by an anti-myc antibody 9E10 and a secondary antibody conjugated to Texas Red. Both phosphatases were localized primarily in the nucleus.



The Expression of VH-1-like PTPases Is Differentially Regulated

We initially compared the distribution of hVH-3 and CL100 mRNAs by Northern blot analysis of human tissues. The distribution of hVH-3 mRNA (2.4 kilobases in length) was limited to the placenta, liver, heart, brain, and kidney, with the highest level of expression occurring in the liver and placenta (Fig. 4A). The pattern of CL100 mRNA expression differed significantly from that of hVH-3 but overlapped in the liver and placenta. Taken together with the distribution of Pac-1, which is primarily limited to hematopoietic cells(7) , these data provide evidence that tissue-specific expression exists among the VH-1-like PTPases. We subsequently tested hepatoma cell lines BRL3, HTC, Reuber H4, and hepG2 cells for the expression of multiple VH-1-like PTPases. hVH-3 was detected in all cells while CL100 mRNA was not measurable. The lack of CL100 expression in four hepatoma cell lines is surprising since the corresponding mRNA is detected from whole liver. The exclusive expression of these two PTPases prohibited direct comparison in cultured cells noted above. However, VHR was co-expressed with hVH-3 in Reuber H4 and HepG2 cells, thus allowing us to compare the response of these two PTPases.


Figure 4: A, distribution of hVH-3 and CL100 mRNA. Northern blot of human tissues was probed for CL100 and hVH-3 mRNA (H, heart; B, brain; PL, placenta; Lu, lung; Li, liver; S, skeletal muscle; Ki, kidney; Pa, pancreas). B, Northern blot analysis of HepG-2 cells. Cells were treated as described under ``Materials and Methods.'' Poly(A) mRNA (5 µg) was blotted and probed for various PTPases. C, Northern blot analysis of Reuber H-4 cells. Cells were treated as described under ``Materials and Methods'' with the exception of dexamethasone (DEX) (1 µM) applied 12 h in advance. Unstim., unstimulated; PMA, phorbol 12-myristate 13-acetate; IGF, insulin-like growth factor; F/IBMX, forskolin/isobutylmethylxanthine; DEX/IL6, dexamethasone/interleukin-6.



Clear differences were observed in the manner in which hVH-3 and VHR respond to extracellular stimuli. For instance, the level of VHR mRNA remained relatively unchanged throughout all treatments (Fig. 4B). In contrast, hVH-3 expression increased in response to several mitogenic stimuli in a cell line-specific manner. Stimulation of HepG-2 cells with either EGF or phorbol 12-myristate 13-acetate for 1 h increased hVH-3 mRNA levels by approximately 5-fold (Fig. 4B). Reuber H4 cells increased hVH-3 expression in response to insulin growth factor-1 and insulin but not to EGF treatment (Fig. 4C). phorbol 12-myristate 13-acetate was effective in both cell lines, suggesting that the signaling pathways that involve the activation of protein kinase C are involved in both cases. Reuber H4 cells are extremely sensitive to insulin stimulation among other hormones, whereas HepG2 cells are responsive to macrophage-derived factors such as interleukins and interferons as well as to growth factors such as EGF. These phenotypic differences may be partially responsible for the observed effects of EGF and insulin growth factor-1 on hVH-3. Other factors, including glucagon and forskolin, that act via the protein kinase A pathway and agents that cause oxidative stress did not influence hVH-3 expression, although these factors have been reported to increase CL100 mRNA levels in other cell types(9, 11) .

The time course of hVH-3 response in Reuber H4 cells to insulin stimulation revealed further differences among the VH-1-like PTPases (Fig. 5). Quantitation of mRNA after correcting for the amount of poly(A) mRNA loaded per lane revealed that hVH-3 mRNA levels increase 15-fold by 30 min while VHR mRNA remain largely unaffected (Fig. 5B). The magnitude and rate of hVH-3 mRNA increase was reminiscent of CL100 expression during serum stimulation(12) . However, unlike CL100, hVH-3 levels remained elevated even after 6 h.


Figure 5: Time course of hVH-3 mRNA increases after stimulation. Reuber H4 cells were stimulated with insulin for the designated length of time. A, the blot was sequentially probed with VHR, hVH-3, and cyclophilin cDNA. B, bands were quantitated densitometrically, normalized to the cyclophilin signal, and expressed as -fold increase over unstimulated levels.



Induction of hVH-3 Expression Is Partially Dependent on Novel Protein Synthesis

The expression of CL100 mRNA during mitotic stimulation of Swiss 3T3 cells is transient, independent of protein synthesis, and enhanced by cycloheximide treatment(8) . To further examine the differences between hVH-3 and CL100 expression, we tested the effect of cycloheximide during stimulation of Reuber H4 cells. Interestingly, we found that addition of cycloheximide did not enhance but instead partially blocked the insulin-induced rise of hVH-3 mRNA (Fig. 6A). The increase in CL100 mRNA content during serum stimulation of Hela cells and its potentiation by cycloheximide treatment is shown for comparison. These data suggest that regulation of hVH-3 during hepatoma cell stimulation differs from that of CL100 and has a component that is dependent on protein synthesis. We suspect that the potentiating effect of cycloheximide on CL100 may be related to the existence of mRNA destabilization signals (27) at the 3`-untranslated region of CL100 (Fig. 6B). Cycloheximide prolongs the half-life of many labile immediate early gene transcripts. The lack of cycloheximide effect on hVH-3 mRNA correlates with the absence of destabilization motifs in the 3`-untranslated region of the mRNA and the apparent stability of its expression during treatments.


Figure 6: Effect of cycloheximide (chx) on mRNA increase. A, increases in hVH-3 mRNA in Reuber H4 cells after stimulated with insulin (100 nM) was compared with those of CL100 mRNA in Hela cells after 20% serum stimulation. Addition of cycloheximide (10 µg/ml) enhanced CL100 mRNA expression but inhibited hVH-3 mRNA levels by approximately 50%. B, the mRNA instability signal AUUUA (underlined) is found repeatedly at the 3`-untranslated region of CL100 mRNA but absent from hVH-3 mRNA. The numbers denote bases from the transcriptional initiation site of CL100.



In this report, we compared the regulation of a novel VH-1-like PTPase with mammalian members of this subfamily. Nuclear localization and in vitro activity toward ERK were features common to this subfamily, leading one to suspect that these PTPases have overlapping functions in vivo. However, these PTPases appear to differ in the duration of mRNA expression, mechanism for activation, and tissue-specific expression. The duration of PTPase expression may directly influence ERK-like kinase activity. Thus, prolonged or transient activation of ERK induced by nerve growth factor or EGF stimulation of PC12 cells, respectively(28) , may involve different VH-1-like PTPases. Finally, it is also likely that multiple dual specificity PTPases are co-expressed in the same tissue and will show selectivity toward a growing family of ERK-like and stress-activated kinases(29, 30) .


FOOTNOTES

*
This work was supported in part by funding from the Walther Cancer Research Institute (Indianapolis, IN) and by Grant 18024 from the NIDDK, National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by 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 GenBank(TM)/EMBL Data Bank with accession number(s) U16996[GenBank].

§
To whom correspondence should be addressed: Minor J. Coon, Professor and Chair, Dept. of Biological Chemistry, Rm. 5416, Medical Science Bldg. 1, The University of Michigan, Ann Arbor, MI 48109-0606. Tel.: 313-764-8192; Fax: 313-763-4581.

(^1)
The abbreviations used are: ERK, extracellular signal-regulated kinase; PTPase, protein-tyrosine phosphatase; EGF, epidermal growth factor; PBS, phosphate-buffered saline; MEK, mitogen-activated protein kinase/extracellular signal-related kinase; VH-1, vaccinia H-1.

(^2)
S. P. Kwak and J. E. Dixon, unpublished observations.


ACKNOWLEDGEMENTS

We thank Drs. C. Worby and R. Stone for helpful suggestions, D. Hakes and K. Martell for their contributions, and Drs. K. L. Guan and E. Butch for providing the ERK and MEK proteins.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.