(Received for publication, November 1, 1994; and in revised form, June 5, 1995)
From the
PTPH1 is a human protein-tyrosine phosphatase with homology to
the band 4.1 superfamily of cytoskeleton-associated proteins. Here, we
report the purification and biochemical characterization of this enzyme
from baculovirus-infected insect cells. The purified protein exhibited
an apparent M of 120,000 on SDS gels. The native
enzyme dephosphorylated both myelin basic protein (MBP) and reduced,
carboxamidomethylated, and maleylated lysozyme (RCML) but was over
5-fold more active on MBP. The K
values
for the two substrates were similar (1.45 µM for MBP and
1.6 µM for RCML). Phosphorylation of PTPH1 by protein
kinase C in vitro resulted in a decrease in K
but had no effect on V
. Removal of the NH
-terminal band
4.1 homology domain of PTPH1 by limited trypsin cleavage stimulated
dephosphorylation of RCML but inhibited its activity toward MBP. The
dephosphorylation of RCML by full-length PTPH1 was enhanced up to
6-fold by unphosphorylated MBP and increasing ionic strength up to 0.2 M NaCl, whereas trypsinized preparations of PTPH1 containing
the isolated catalytic domain were unaffected. These results suggest
that in addition to a potential role in controlling subcellular
localization, the NH
-terminal band 4.1 homology domain of
PTPH1 may exert a direct effect on catalytic function.
Reversible protein phosphorylation on tyrosyl residues is an
essential feature of the regulation of many cellular functions and is
controlled by the opposing action of protein-tyrosine kinases and
protein-tyrosine phosphatases (PTPs). ()PTPs comprise a
large and growing family of receptor and cytoplasmic signaling enzymes
sharing a conserved catalytic domain of 240 residues containing the
signature motif
(I/V)HCXAGXXR(S/T)G(1, 2) . The
catalytic domains of different PTPs are flanked by a variety of
noncatalytic segments. The NH
-terminal extracellular
segments of receptor PTPs may transduce a signal across the plasma
membrane through their specific response to different ligands. The
noncatalytic segments of cytoplasmic PTPs contain domains homologous to
motifs such as SH2 domains for binding to phosphotyrosine, band
4.1-related domains for targeting to interfaces between the plasma
membrane and cytoskeleton and domains for potential lipid
binding(3) . These domains not only may be involved in
compartmentalization of PTPs but also may directly modulate catalytic
activity or recognition of substrates.
PTPH1 was isolated as a
putative intracellular PTP from a HeLa cell cDNA library(4) .
PTPH1 and PTPMEG1(5) , as well as the recently identified
PTP-BAS/1E/L1 (6, 7, 8) and
PTPD1/RL10/2E(9, 10, 11) , constitute an
expanding subfamily of PTPs that contain NH-terminal
segments of homology to cytoskeleton-associated proteins of the band
4.1 superfamily that includes band 4.1, ezrin, talin, radixin, moesin,
and merlin. The catalytic domain of PTPH1 is contained in the
COOH-terminal portion of the molecule separated from the
NH
-terminal, band 4.1-related domain by a central segment
that contains a GLGF (or DHR) sequence motif (residues 511-596,
30% identity and 57% similarity to GLGF-repeat I in the Drosophila tumor suppressor discs-large (Dlg)) (12) and a
potential SH3 binding site (residues 69-77, PSRSPPITP). The
function of GLGF motifs is currently unknown, but in addition to the
band 4.1-related PTPs, they have been found in a rapidly expanding
family of intracellular proteins such as erythrocyte palmitoylated
protein p55, nitric oxide synthase, postsynaptic density protein
Psd-95, and tight junction protein ZO-1(13) . Recently, the
GLGF region from human Dlg was shown to bind the amino-terminal domain
of protein 4.1(14) . Band 4.1 homology domains are responsible
for targeting cytoskeleton-associated proteins to cytoskeleton-membrane
interfaces (15) , as illustrated in the association of band 4.1
with glycophorin and p55 (16) and that of moesin with
CD44(17) . The presence of such a targeting domain implies that
PTPH1 may be also directed to cytoskeleton-membrane interfaces and that
the substrate selectivity of PTPH1 may be determined, at least in part,
by its subcellular localization. If so, cytoskeleton-associated
proteins such as band 4.1 or ezrin, tyrosine phosphorylation of which
correlates with their redistribution into the
cytoskeleton(18, 19, 20) , may be typical
substrates of this enzyme in vivo. Interestingly, tyrosine
phosphorylation of several cytoskeleton- and membrane-associated
proteins such as ezrin has been linked to malignant
transformation(21, 22) .
We have expressed PTPH1 in baculovirus-infected insect cells and demonstrated that it possesses intrinsic enzymatic activity. We have observed that PTPH1 displays substrate selectivity, and this is conferred at least in part by the noncatalytic band 4.1-homology domain. Thus, in addition to the potential for controlling subcellular distribution, the band 4.1 domain may exert a direct effect on the catalytic activity of PTPH1.
Figure 1: Purification and detection of PTPH1. A, elution profile of PTPH1 from FFQ-Sepharose. The plain curve represents absorbance at 280 nm, the filled dots represent PTP activity, and the dashed line represents the NaCl gradient from 0-0.5 M. Fractions 27-36 were pooled for purification on Mono-S-Sepharose. B, elution profile of PTPH1 from Mono-S. The dashed line represents the gradient of NaCl, 0-0.8 M. The other symbols are as described for A. The peak of enzyme activity eluted at 0.74 M NaCl (fractions 48-49). PTP assays were performed with 3 µM RCML as substrate in both A and B. C, SDS-PAGE analysis of peak fractions throughout the purification procedure (lysate, 10 µg; FFQ fraction, 4 µg; Mono-S fraction, 0.4 µg) stained with Coomassie Blue. The molecular mass standards are indicated in kilodaltons on the left.
Figure 2:
Phosphorylation of PTPH1. A,
autoradiogram showing phosphorylation of PTPH1 by various protein
kinases. PTPH1 (1.9 µg) was phosphorylated using 0.1 µg of
protein kinase as described under ``Experimental
Procedures.'' After 30 min, phosphorylation was terminated by
addition of an equal volume of 2 SDS sample buffer, and the
mixture was subjected to SDS-PAGE. PKA, protein kinase A; PKC, protein kinase C; CKII, casein kinase II; MAPK, MAP kinase. B, autoradiogram showing that PTPH1
is a phosphoprotein in vivo. PTPH1 was immunoprecipitated from
P-labeled 293 cells using a polyclonal antiserum and
subjected to SDS-PAGE. Both above gels were dried and exposed to
autoradiography. Molecular weight standards are indicated on the left. mIgG, normal mouse IgG; pAbZ5,
polyclonal anti-PTPH1 serum.
Figure 3: Effect of trypsinization on enzymatic activity of PTPH1. PTPH1 (0.19 µg, which following dilution from the purification buffer still contained protease inhibitors 0.1 µg/ml aprotinin, 0.1 µg/ml leupeptin, and 0.1 mM benzamidine) was exposed to trypsin (0.19 µg) at 30 °C for 5 min. Trypsin was inactivated with lima bean trypsin inhibitor, and the PTPH1 was assayed in duplicate using phosphotyrosyl MBP and RCML as substrates. Data points from the lowest RCML concentrations tested have been omitted from the graph for clarity, but were included in the analysis that generated the regression lines. The initial velocity (V) is expressed as nmol/min, and the concentration of substrates (S) is in µM. Circles, RCML as substrate; triangles, MBP as substrate; open symbols, PTPH1 not exposed to trypsin; filled symbols, PTPH1 exposed to trypsin.
To correlate the appearance of particular proteolytic products with
enzyme activity, a time course of trypsinization was performed, and the
effect on activity against RCML was determined by enzyme assays (Fig. 4A). At each time point, the proteolytic products
were also analyzed by SDS-PAGE. As shown in Fig. 4, B and C, a fragment of 50 kDa containing the catalytic
domain was first generated following trypsinization; however, peak
activation of PTPH1 toward RCML (8 min) correlated with the appearance
of three tryptic peptides of 39 kDa, 37 kDa, and 36.5 kDa (p39, p37,
and p36.5 in Fig. 4B, inset). Immunoblotting
with a monoclonal antibody to the catalytic domain of PTPH1 (Fig. 4C) indicates that of these species, the p39
fragment contains the catalytic domain and its appearance coincides
with maximal activation. Microsequencing of p39 indicates an
NH
-terminal sequence of SFADFKSEDELNQLFP, which is
identical to that beginning at residue 601, the start of the catalytic
domain of PTPH1 (Table 2). A fragment comprising residue 601 to
the COOH terminus (residue 913) would represent 313 residues,
predicting an M
of 36,000. Thus, p39 most likely
represents the intact COOH-terminal segment of PTPH1 containing its
catalytic domain. The fragments p37 and p36.5 display identical
NH
-terminal sequences of TRSEVICSIHFL corresponding to the
segment of PTPH1 beginning at residue 25 (Table 2). This
represents the predicted NH
terminus of the band 4.1
homology domain. The apparent M
of these peptides
on SDS-PAGE is in close agreement to the calculated size of the band
4.1 domain (38,500), thus they most likely represent the intact domain.
Interestingly, this domain appears more resistant to trypsin than the
catalytic domain. Apparently, most of the trypsin-sensitive sites are
in the central segment, which is unique to PTPH1.
Figure 4:
Time course of trypsin cleavage and
activation of PTPH1 toward RCML. Equal amounts of PTPH1 (containing
proteinase inhibitors as in Fig. 3) and trypsin were mixed and
incubated at 30 °C. At the times indicated, samples were taken and
lima bean trypsin inhibitor was added in a 60-fold excess by weight.
Samples were then divided for analysis by SDS-PAGE or for PTP assay. A, time course of activation of PTPH1 by trypsinization.
Phosphotyrosyl RCML was used as the substrate. Each time point is the
average of duplicate assays. Each PTP assay contains 0.1 µg of
purified PTPH1. B, resolution of trypsinized fragments of
PTPH1 by 10% SDS-PAGE. The gel was stained with Coomassie Blue. Inset, silver stain of indicated (boxed) area in B (p39 was stained better with silver). The bars indicate the fragments (p39, p37, and p36.5) whose NH termini were sequenced. The appearance and disappearance of p39
correlated with the activation and inactivation of PTPH1. C,
analysis of trypsinization of PTPH1 by immunoblotting with QY8, the
monoclonal antibody to the catalytic
domain.
Following tryptic cleavage,
to release the free catalytic domain, the dephosphorylation of RCML by
PTPH1 was stimulated 5-10-fold, suggesting the possibility that
in the full-length protein the catalytic domain may be subjected to an
inhibitory constraint. In light of the fact that the V for dephosphorylation of MBP was 7-fold higher than for RCML, we
tested whether the effect of proteolytic activation on the
dephosphorylation of RCML could be mimicked by adding various agents,
including MBP. As shown in Fig. 5A, addition of MBP at
increasing concentrations up to 20 µM stimulated the
activity of full-length PTPH1 toward RCML up to 6-fold. Similarly,
increasing the salt concentration up to 0.1-0.2 M NaCl
activated PTPH1 toward RCML as substrate by nearly 6-fold (Fig. 5B). The same concentration of either MBP or NaCl
that stimulated dephosphorylation of RCML by full-length PTPH1 had no
effect on the activity of the isolated catalytic domain generated by
trypsin cleavage. Similar results were observed with the polycarboxylic
acids EDTA, EGTA, and tartrate (Table 3). Therefore, the effects
of these charged species were apparently mediated by the noncatalytic
NH
-terminal segment of PTPH1.
Figure 5: Activation of PTPH1 toward RCML as substrate by MBP (A) and NaCl (B). Activity of PTPH1 (0.1 µg) was assayed with 8.7 µM phosphotyrosyl RCML in the presence of the indicated concentration of unphosphorylated MBP or NaCl. Fold activation of PTPH1 was calculated by comparison with the activity of PTPH1 in control low ionic strength assay buffer. Solid circles indicate full-length PTPH1. Open triangles represent trypsin-truncated PTPH1.
Protein-tyrosine phosphorylation plays a crucial role in the regulation of a dynamic cytoskeleton in normal cell growth(15, 19, 20) . Although the tyrosine phosphorylation of cytoskeletal components may be catalyzed by protein-tyrosine kinases such as focal adhesion kinase and c-src that are associated with membranes and the cytoskeleton(27) , dephosphorylation would require the presence of PTPs in the same subcellular compartments. PTPH1, which displays homology to the cytoskeleton-associated proteins of the band 4.1 superfamily, may be an important regulator of cytoskeleton integrity. As an initial step to characterizing the function of PTPH1, we have purified the enzyme from Sf9 cells infected by PTPH1-expressing baculovirus.
PTPH1 purified to apparent homogeneity efficiently
dephosphorylated two tyrosine-phosphorylated substrates, MBP and RCML, in vitro. The native enzyme displayed a 7-fold higher V against MBP than against RCML, consistent with
a degree of substrate selectivity in vitro. In addition, PTPH1
activity could be specifically blocked by PTP inhibitors but not by
inhibitors of serine/threonine, alkaline, or acid phosphatases. Thus,
these results clearly establish that PTPH1 is a protein-tyrosine
phosphatase.
Because the central segment of PTPH1 that divides the band 4.1-related and catalytic domains contains clusters of consensus phosphorylation sites, phosphorylation may be of significance in regulating PTPH1 in vivo. We have observed that PTPH1 is phosphorylated in vitro to high stoichiometry (2 mol/mol) by protein kinase C with a concomitant increase in activity due to a phosphorylation-induced increase in affinity of the enzyme for substrate. In addition, PTPH1 is a phosphoprotein in vivo, phosphorylated on seryl and threonyl residues; however, the protein kinase responsible for the modification remains to be identified. Purified PTPH1 immigrated as a doublet upon SDS-PAGE, but treatment with acid phosphatase, alkaline phosphatase, or PP2A did not affect its mobility. Thus the nature of the modification that gives rise to the appearance of the doublet is still unclear.
Several examples now point to the importance of subcellular compartmentalization facilitated by protein-protein interactions as a strategy for regulation of PTP function(1) . By analogy with the interaction between band 4.1 and glycophorin and the interaction of ezrin, radixin, and moesin with CD44, it is likely that PTPH1 interacts via its band 4.1-related domain with the cytoplasmic segments of other transmembrane proteins. In addition, the central segment of PTPH1 containing the GLGF motif and the putative SH3 domain binding motif may interact with other proteins. Furthermore, individual band 4.1 superfamily members such as ezrin and moesin have been shown both to bind homotypically and to form heterotypic associations with other members of the family(28, 29) . Thus, such interactions involving PTPH1 may lead to the translocation and anchorage at the cytoskeleton-membrane interfaces and play a role in regulating cytoskeletal organization or transmembrane signaling.
In addition to
its potential role for targeting and interacting with other proteins,
we have demonstrated that the NH-terminal segment may also
directly regulate the enzymatic activity of PTPH1. Proteolytic removal
of the noncatalytic segment of PTPH1 increased its PTP activity in
vitro toward RCML by up to 10-fold, similar to the case of T-cell
PTP, where a 30-fold increase in PTP activity toward RCML was observed
upon trypsinization(30) . The activity toward RCML of
full-length PTPH1 but not the isolated catalytic domain was also
stimulated in vitro by increasing ionic strength or by adding
charged species including MBP or polycarboxylic acids. These
observations suggest that charged molecules may attenuate interactions
between the noncatalytic segment of PTPH1 and the catalytic domain.
Similar results has been obtained with the SH2 domain-containing PTPs (31, 32) . This situation is reminiscent of molecules
such as protein kinase C and myosin light chain kinase(33) , as
well as the protein serine/threonine phosphatase
calcineurin(34) . In all these cases, noncatalytic segments on
either the NH
- or COOH-terminal side of the catalytic
domain exert an inhibitory effect on enzymatic activity in the basal
state. These segments contain autoinhibitory or pseudosubstrate motifs
that possess features of the normal substrate for the enzyme and occupy
the active site under basal conditions. Binding of
Ca
/calmodulin to myosin light chain kinase or
calcineurin or Ca
/phospholipids to protein kinase C
induces a conformational change in the enzyme in which the
autoinhibitory segment is removed from the active site, which is thus
opened for catalysis. Limited proteolytic digestion of these enzymes
has been shown to cleave off the autoinhibitory sequences to generate a
truncated enzyme with high basal activity that is no longer dependent
upon binding of the allosteric effectors. Therefore, we propose that
the noncatalytic, NH
-terminal segment of PTPH1 exerts an
inhibitory effect on catalytic function and that association with
regulatory/targeting proteins may release this inhibition in vivo following the paradigm of the examples described above.