©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Biochemical Characterization of a Human Band 4.1-related Protein-tyrosine Phosphatase, PTPH1 (*)

(Received for publication, November 1, 1994; and in revised form, June 5, 1995)

Shao-Hui Zhang (§) William R. Eckberg (¶) Qing Yang (**) Ahmed A. Samatar Nicholas K. Tonks (§§)

From the Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2208

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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(r) 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(max). Removal of the NH(2)-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(2)-terminal band 4.1 homology domain of PTPH1 may exert a direct effect on catalytic function.


INTRODUCTION

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). (^1)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(2)-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(2)-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(2)-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.


EXPERIMENTAL PROCEDURES

Expression and Purification of PTPH1

Sf9 cells were infected with recombinant baculovirus expressing PTPH1, generated using the Baculogold system (Pharmingen, San Diego, CA) with a construct obtained by ligating the full-length PTPH1-cDNA (4) into the EcoRI site of vector pVL1393 (Invitrogen, San Diego, CA). Cells were harvested 72 h postinfection and homogenized by 15 strokes of a Dounce homogenizer in 5 volumes of lysis buffer (20 mM Tris-HCl, pH 7.5, 5 mM EDTA, 2 mM DTT, 5 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM benzamidine). Homogenates were clarified by centrifugation at 125,000 g for 20 min. The supernatant was loaded onto a FFQ-Sepharose column (10 1 cm) equilibrated with 20 mM Tris-HCl, pH 7.5, 1 mM DTT, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 mM benzamidine and eluted with a linear gradient of 0-0.5 M NaCl in the same buffer. Active fractions were pooled, dialyzed overnight against 50 mM Hepes, pH 7.5, 1 mM DTT, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 1 mM benzamidine, loaded on a Mono-S column (5 0.5 cm), and eluted with a linear gradient of 0-0.8 M NaCl in the same buffer.

Assay of PTPH1 Activity

PTP activity was assayed as described previously (23) using tyrosine-phosphorylated substrates MBP and RCML. To test the effect of trypsinization on activity, PTPH1 was incubated at 30 °C with trypsin (at a weight ratio of PTPH1:trypsin, 1:1) in a buffer of 25 mM imidazole-HCl, pH 7.5, 1 mM DTT, 0.1 µg/ml aprotinin, 0.1 µg/ml leupeptin, and 0.1 mM benzamidine for various lengths of time. The high concentration of trypsin was used to overcome the protease inhibitors in the PTPH1 preparation. After incubation, trypsin was inactivated with a 60-fold excess by weight of lima bean trypsin inhibitor. Trypsin-treated PTPH1 was then assayed for activity and subjected to SDS-PAGE followed by immunoblotting to examine the extent of proteolysis. For NH(2)-terminal sequence analysis, PTPH1 was trypsinized for 8 min, and peptides were fractionated by SDS-PAGE and transferred to polyvinylidene difluoride membrane. Proteolytic fragments were excised from the membrane after staining with Ponceau S and sequenced on an ABI automatic protein sequencer 473 (Foster City, CA).

Antibody to PTPH1 and Immunoblotting

A glutathione S-transferase-PTPH1 fusion protein containing the catalytic domain (residues 633-913) was expressed in Escherichia coli, purified by preparative SDS-PAGE, and used to generate monoclonal antibody QY8. A polyclonal anti-PTPH1 serum, pAbZ5, was generated in mouse using purified full-length PTPH1 from Sf9 cells as antigen. Immunoblot analysis was performed using an enhanced chemiluminescence protocol as described previously(24) .

Phosphorylation of PTPH1

Purified PTPH1 was incubated with MAP kinase (p42)(25) , the catalytic subunit of protein kinase A (Sigma), protein kinase C (A. Nairn, Rockefeller University), cdc2, and casein kinase II (D. Marshak, Cold Spring Harbor Laboratory). Reactions were carried out in kinase buffer containing 10 mM Hepes, pH 7.5, 10 mM MgCl(2), 1 mM DTT, and 100 µM [-P]ATP (5,000 cpm/pmol). 50 µg/ml phosphatidylserine, 5 µg/ml diacylglycerol, and 1 mM CaCl(2) were added to the protein kinase C assays; 150 mM NaCl was added to the casein kinase II assays. Phosphorylation was terminated by boiling in SDS sample buffer. After electrophoresis, gels were fixed, dried, and exposed to x-ray film. PTPH1 bands were excised from the gel, and their radioactivity was quantitated by liquid scintillation counting. To test the effect of phosphorylation of PTPH1 on its activity, parallel reactions were conducted without radioactive ATP. The PTPH1 activity was then assayed as described and compared with that of PTPH1 that had been incubated with protein kinase C in the absence of ATP.

In Vivo Labeling and Immunoprecipitation

Subconfluent 293 cells were incubated with 0.5 mCi/ml [P]orthophosphate (ICN Radiochemicals, Irvine, CA) at 37 °C for 9 h in phosphate-free Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. Cells were washed in phosphate-buffered saline, lysed in Nonidet P-40 lysis buffer and clarified as described previously(26) . 1 mg of cell lysate was precleared for 1 h with 20 µg of normal mouse IgG bound to protein A-Sepharose beads in 1 ml of Nonidet P-40 buffer. The beads were collected by centrifugation, and the supernatant was incubated with 5 µl of pAbZ5 bound to protein A-Sepharose for 2 h. The immunocomplexes were washed 5 times with Nonidet P-40 buffer and analyzed by SDS-PAGE and autoradiography.


RESULTS

Purification of PTPH1

The level of expression of PTPH1 in baculovirus-infected Sf9 cells was determined by immunoblotting of cell lysates at different times postinfection. At 72 h postinfection, 80% of PTPH1 protein is recovered in the soluble fraction extracted with aqueous lysis buffer (data not shown). The purification of PTPH1 from infected Sf9 cells is illustrated in Fig. 1. The protein eluted from FFQ-Sepharose at 0.35 M NaCl (Fig. 1A) and from Mono-S at 0.74 M NaCl (Fig. 1B). PTPH1 from the Mono-S column was observed to migrate as a doublet, with an apparent M(r) of 120,000 by SDS-PAGE (Fig. 1C). This is consistent with the size of PTPH1 polypeptide produced in a reticulocyte lysate using PTPH1 mRNA synthesized from the cDNA or PTPH1 detected by immunoblotting lysates of human A431 or 293 cell lines (data not shown). Following this procedure, PTPH1 was purified 18-fold from infected Sf9 cells with a 17% yield (Table 1). Thus, PTPH1 constitutes 5% of the total protein of the baculovirus-infected cells.


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.





Kinetic Properties of PTPH1

PTPH1 dephosphorylated tyrosine-phosphorylated preparations of MBP and RCML, but showed different activity against the two substrates. Although the K for both substrates is very similar (1.6 µM for RCML and 1.45 µM for MBP), the V(max) for the two substrates differed substantially. The V(max) for MBP is 1493 nmol/min/mg and that for RCML is 220 nmol/min/mg. These values are within the expected range for members of the PTP family. The purified PTPH1 displayed a broad pH optimum over the range 6.0-8.0.

Phosphorylation of PTPH1

PTPH1 was phosphorylated to different levels by protein serine/threonine kinases protein kinase A, protein kinase C, and MAP kinase in vitro (Fig. 2A). Addition of 1 mM Na(3)VO(4) to the MAP kinase reaction did not affect the phosphorylation of PTPH1 by MAP kinase, suggesting that PTPH1 does not dephosphorylate and inactivate MAP kinase under these conditions. A 30-min incubation with protein kinase C resulted in phosphorylation to a stoichiometry of 2 mol/mol, the most extensive phosphorylation observed in our studies. Following phosphorylation by protein kinase C, PTPH1 displayed increased activity (by 25%) at the lowest substrate concentrations tested, although the V(max) was unaffected. Thus phosphorylation marginally enhanced the affinity of PTPH1 for its substrate. As shown in Fig. 2B, PTPH1 was observed to be a phosphoprotein in vivo in human 293 cells. Phosphorylation occurred predominantly on seryl residues, although some phosphorylation of threonyl residues was also detected (data not shown).


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.



Proteolytic Activation of PTPH1

To test the possibility that the noncatalytic segment of PTPH1 may exert an effect on the activity of the enzyme, a series of limited trypsin digests were performed. As shown in Fig. 3, trypsinization of PTPH1 affected its activity on the two substrates differently. Dephosphorylation of RCML could be stimulated 5-10-fold by trypsinization, whereas that of MBP was inhibited by 30%. For example, in one set of experiments, after trypsinization the V(max) for MBP decreased from 1493 to 1067 nmol/min/mg, whereas that for RCML increased from 213 to 826 nmol/min/mg. In both cases, the K values were decreased, for RCML from 1.60 to 0.78 µM and for MBP from 1.45 to 0.62 µM. These results suggest that the presence of an intact noncatalytic segment in PTPH1 can affect its substrate preference.


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(2)-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(r) 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(2)-terminal sequences of TRSEVICSIHFL corresponding to the segment of PTPH1 beginning at residue 25 (Table 2). This represents the predicted NH(2) terminus of the band 4.1 homology domain. The apparent M(r) 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(2) 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.





Effectors of PTPH1 Activity

To identify modulators of PTPH1, a battery of potential effectors were tested (Table 3). As expected, the classical PTP inhibitors vanadate, molybdate, zinc, and tungstate inhibited the activity of PTPH1 by >90%. The protein serine/threonine phosphatase inhibitors okadaic acid and NaF, the alkaline phosphatase inhibitor tetramisole, the acid phosphatase inhibitor tartrate, and divalent cations such as calcium and magnesium did not inhibit the activity of PTPH1.



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(max) 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(2)-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.




DISCUSSION

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(max) 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(2)-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(2)- 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(2)-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.


FOOTNOTES

*
This work was supported by Grant CA53840 from the National Cancer Institute. 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.

§
Cold Spring Harbor Association Fellow.

Supported by the Council for Tobacco Research, U.S.A. Inc. Present address: Dept. of Biology, Howard University, Washington, D. C. 20059.

**
Present address: Gene Therapy Center, 944 Wilson Hall CB#7352, University of North Carolina, Chapel Hill, NC 27599.

§§
Pew Scholar in the Biomedical Sciences. To whom correspondence should be addressed: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724-2208. Tel.: 516-367-8846; Fax: 516-367-6812; Tonks{at}cshl.org.

(^1)
The abbreviations used are: PTP, protein-tyrosine phosphatase; DTT, dithiothreitol; FFQ, Fast Flow Q; MAP, mitogen-activated protein; MBP, myelin basic protein; RCML, reduced, carboxamidomethylated, and maleylated lysozyme; PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Dr. Ryuji Kobayashi for peptide sequencing and Carmelita Bautista for generating monoclonal antibodies. We also thank Drs. A. J. Flint and R. L. Del Vecchio for critical comments.


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