(Received for publication, September 8, 1994; and in revised form, November 11, 1994)
From the
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.
Extracellular signal-regulated kinase (ERK) ()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) . ()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.
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.
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
VO
(lane4, ERK alone; lane5, hVH-3 (20 ng)
+ NaF (100 mM), NaPPi (2 mM); lane6, hVH-3 (20 ng) + Na
VO
(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.
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.
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) .
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) U16996[GenBank].