©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Receptor-like Protein-tyrosine Phosphatase, RPTP, Is Phosphorylated by Protein Kinase C on Two Serines Close to the Inner Face of the Plasma Membrane (*)

Sharon Tracy (§) , Peter van der Geer (¶) , Tony Hunter (**)

From the (1) Molecular Biology and Virology Laboratory, The Salk Institute, La Jolla, California 92037

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To determine whether the receptor-like protein-tyrosine phosphatase, RPTP, which is widely expressed in both the developing and adult mouse, is regulated by phosphorylation, we raised antiserum against a C-terminal peptide. This antiserum precipitated a 140-kDa protein from metabolically S-labeled NIH3T3 cells. Using this antiserum, we showed that endogenous RPTP is constitutively phosphorylated in NIH3T3 cells, predominantly on two serines, which we identified as Ser-180 and Ser-204, lying in the juxtamembrane domain. 12- O-tetradecanoylphorbol-13-acetate (TPA) stimulation of quiescent NIH3T3 cells rapidly increased phosphorylation of Ser-180 and Ser-204. Purified protein kinase C (PKC) phosphorylated bacterially expressed RPTP at Ser-180 and Ser-204. When wild type and S180A/S204A double mutant RPTPs were transiently expressed in 293 human embryonic kidney cells, TPA stimulated phosphorylation of wild type but not of double mutant RPTP. PKC down-regulation following prolonged exposure to TPA diminished TPA-stimulated RPTP phosphorylation. Taken together, these results indicate that RPTP is a direct substrate for (PKC). Examination of 293 cells expressing exogenous RPTP using immunofluorescence confocal microscopy showed that RPTP exists predominantly in two subcellular compartments: in dense intracellular granules or dispersed within the plasma membrane. TPA treatment caused redistribution of some intracellular RPTP to the cell surface, but this did not require direct phosphorylation of RPTP at Ser-180/Ser-204. Our results suggest that activation of PKC by cytokines modulates RPTP function in several different ways.


INTRODUCTION

Signal transduction by protein-tyrosine kinases is relatively well understood, but rather little is known about their enzymatic counterpart, the protein-tyrosine phosphatases (PTPs).() Over the past few years, a large number of PTPs have been cloned, based on sequence homology within the catalytic domains (for reviews, see Refs. 1-4). Broadly speaking, there are two types of PTP: membrane-spanning receptor-like PTPs and nonreceptor PTPs. Most but not all receptor-like PTPs (RPTPs) contain two cytoplasmic catalytic PTP domains. The domain closest to the membrane exhibits the majority of the PTP activity, but in some cases the second domain also possesses detectable but low PTP activity (5, 6) . RPTP extracellular domains range in size from over 1600 to 27 residues. Whether the extracellular domain of RPTPs can bind ligands and, if they do, whether this modulates PTP activity remains to be determined.

RPTP (also known as HRPTP (7) , HPTP (8) , LRP (LCA-related phosphatase) (9) , HLRP (10) , and R-PTP- (11) ) is a RPTP, with a rather short extracellular domain lacking any obvious structural motifs or cysteines (1, 2) . Mammalian RPTP contains 793 amino acids, including a 19-residue signal sequence, a 123-amino acid extracellular domain, a transmembrane domain, two catalytic domains, and a short C-terminal tail. Both catalytic domains have been shown to have in vitro PTP activity when expressed individually in bacteria (5) or collectively when expressed in insect cells (12) . RPTP is most highly expressed in brain, both in embryonic development and in the adult (11) . The fact that RPTP mRNA expression is enhanced during differentiation of neuronally derived cell lines suggests that RPTP may be involved in neuronal differentiation. This idea is reinforced by the finding that overexpression of RPTP in pluripotent P19 embryonal carcinoma cells alters the differentiation fate of these cells in favor of neuronal differentiation (13) . The nonreceptor protein-tyrosine kinase c-Src is activated in the RPTP-overexpressing P19 cells (13) and in RPTP-overexpressing Rat-2 cells (14) , possibly as a result of direct dephosphorylation of the c-Src inhibitory tyrosine phosphorylation site (Tyr-527) by RPTP.

Relatively little is known about regulation of PTP activity, but there are indications that both serine and tyrosine phosphorylation may play an important role. Treatment of T cells with the calcium ionophore ionomycin leads to a decrease in CD45 activity concomitant with a decrease in CD45 serine phosphorylation (15) . Serine phosphorylation of CD45 and of the cytoplasmically localized PTP1B is enhanced in response to phorbol ester treatment (16, 17) . However, these phosphorylation events do not significantly affect the activity of either PTP. In contrast, phorbol ester treatment of HL-60 cells has been found to increase the expression, activity and serine phosphorylation of PTP1C and its translocation to the plasma membrane (18) , but it is not clear that this phosphorylation is directly due to PKC nor whether it affects PTP1C activity indirectly. TPA treatment also leads to a small increase in RPTP activity, concomitant with an increase in serine phosphorylation, and in this case phosphorylation has been shown to decrease the Kfor phosphopeptide substrate (19) . PTP-PEST is phosphorylated in vitro and in vivo by PKC, but in this case its activity is decreased due to an increase in its Kfor substrate (20) . It has been shown that MAP kinase phosphorylates and inactivates PTP2C (also known as PTP1D, Syp, and SH-PTP2) in vitro, and this phosphorylation may be responsible for the inhibition of PTP2C observed in EGF-treated PC12 cells during the time when MAP kinase is activated (21) .

There are also a number of reports of tyrosine phosphorylation of PTPs. The SH2-containing nonreceptor PTP, PTP1D, is phosphorylated on tyrosine in response to activation of the EGF and platelet-derived growth factor receptor-like protein-tyrosine kinases (22, 23) , but whether tyrosine phosphorylation affects PTP1D activity remains to be established definitively. CSF-1 treatment of mouse macrophages induces tyrosine phosphorylation of the related SH2-containing PTP, PTP1C (24) . We have found that RPTP is constitutively phosphorylated on Tyr-789 in fibroblasts, and that protein Tyr-789 acts as a binding site for the adaptor protein Grb2 (25) . Su et al.(26) have also shown that RPTP is tyrosine phosphorylated and associates with Grb2. RPTP CD45 is phosphorylated on tyrosine in vivo following treatment of cells with the PTP inhibitor phenylarsene oxide (27) , but whether treatment affects activity in vivo is unclear. The same group has recently reported that the PTP activity of RPTP CD45 is increased in vitro by sequential phosphorylation first by a protein-tyrosine kinase and second by a protein-serine kinase (28) . The order of phosphorylation is critical, and the increase in activity is also specific for the RCML substrate. Proteolytic removal of the first catalytic domain produces a similar activation (6) , implying that the increase in RCML-specific PTP activity is due to the second catalytic domain, consistent with the sites of phosphorylation being located within the N-terminal portion of the second catalytic domain.

To begin to characterize RPTP function, we asked whether it is phosphorylated in vivo and whether its phosphorylation can be modulated in response to growth factors. We have shown that RPTP is constitutively phosphorylated predominantly on serine and have located the two serine residues close to the inner face of the plasma membrane as major sites of phosphorylation. Phorbol ester treatment increases phosphorylation at these sites, and purified PKC phosphorylates these same serines in vitro.


MATERIALS AND METHODS

Cloning PTP and Construction of Recombinant bPTP

Sequences for the PTPs PTP1B (29) , CD45 (30) , and LAR (31) had been reported at the onset of this investigation. These sequences were aligned, and regions conserved in the three PTPs were used to design oligonucleotide primers specific for the catalytic domain. polymerase chain reactions were performed using RNA from NIH3T3 cells as a template. The product of this reaction was used to probe an NIH3T3 cDNA library. A clone encoding a protein with a structure typical of a CD45-like phosphatase was isolated. This clone, pPTP, was used in all subsequent experiments. The sequence and predicted translation product of pPTP are essentially identical to other reported mouse RPTP clones (9, 11) . Recombinant PTP was generated by fusing the cytoplasmic domain of PTP beginning at Phe-168 to the glutathione S-transferase gene of the expression plasmid pGEXKG (pGEXPTP). The fusion protein was purified on glutathione-Sepharose beads and cleaved with thrombin generating bPTP.

Cell Culture, Labeling, and Transient Transfection

For P labeling, NIH3T3 cells were grown to 75% confluence in Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum (CS) and then shifted to DMEM containing 0.5% CS. After 24 h, the cells were labeled in phosphate-free DMEM containing 0.5% dialyzed CS and 2 mCi/ml [P]orthophosphate (ICN) for 16 h at 37 °C. Cells were stimulated by the addition of 50 ng/ml TPA (Sigma) for 15 min at 37 °C. For S labeling, 2 10 NIH3T3 cells grown under the same conditions were labeled with 100 µCi/ml [S]methionine/cysteine (Express, DuPont NEN) for 20 h at 37 °C in methionine-free DMEM containing 4% dialyzed CS. For transfection experiments, human embryonic kidney 293 cells were seeded on plates coated with 0.1% gelatin in DMEM containing 10% calf serum. 24 h later, cells were transfected with 20 µg of Sg5 vector, which utilizes a SV40 promoter to drive expression, containing either full-length wild type (13) or S180A/S204A mutant RPTP. Transfections were done using the calcium phosphate method as previously described (25) . 8 h after transfection, the medium was changed to either methionine-free DMEM containing 1% dialyzed CS or phosphate-free DMEM containing 1% dialyzed CS and incubated an additional 16 h at 37 °C. Cells were stimulated by the addition of 100 ng/ml TPA for 10 min at 37 °C (initial time course experiments had shown that the shorter time was optimal for the transfected cells).

Antiserum and Immunoprecipitation

Polyclonal rabbit antiserum, 5201, was raised against the peptide CYKVVQEYIDAFSDYANFK corresponding to the C-terminal 19 residues of RPTP. The peptide was coupled to KLH (Calbiochem) and used to immunize rabbits (32) . Polyclonal rabbit antiserum, 5478, was raised against purified bPTP protein and purified on a GST-bPTP affinity column (25) . S- or P-labeled cells were lysed in SDS-boiling lysis buffer (10 mM sodium phosphate, pH 7.2, 0.5% SDS, 1% aprotinin, 1 mM EDTA, 1 mM dithiothreitol (DTT)), boiled, and diluted with 4 volumes of RIPA buffer (10 mM sodium phosphate, pH 7.0, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM DTT but lacking SDS. Lysates were prepared and proteins were immunoprecipitated with 8 µl of preimmune serum, 8 µl of immune serum, or 8 µl of immune serum preincubated with 10 µg of immunizing peptide. The immunoprecipitates were collected on protein A-Sepharose and washed four times in RIPA buffer without DTT. The labeled proteins were resolved on a 7.5% SDS-polyacrylamide gel and transferred electrophoretically to Immobilon-P or nitrocellulose membranes.

Expression, Purification, and in Vitro Phosphorylation of Recombinant bPTP

Expression, purification, and thrombin cleavage of bPTP were performed as described (33) except that cells were lysed by sonication in PBS containing 1% Triton X-100 (Sigma), 1% aprotinin, and 1 mg/ml lysozyme. Purified rat brain PKC was the gift of Ushio Kikkawa and was prepared as described (34) . bPTP was phosphorylated in vitro by purified PKC under the following reaction conditions: 20 mM Hepes, pH 7.4, 10 mM MgCl, 0.5 mM CaCl, 5 mM DTT, 8 µg/ml phosphatidylserine (Sigma), 0.8 µg/ml diolein (Avanti), 100 ng of purified PKC, and 25 µCi of [-P]ATP (3000 Ci/mmol, Amersham Corp.). The reaction was incubated for 15 min at 30 °C and was terminated by the addition of EDTA to a final concentration of 5 mM. Sample buffer was added, and the samples were then boiled and resolved on a 10% SDS-polyacrylamide gel and transferred to nitrocellulose.

Phosphoamino Acid Analysis and Two-dimensional Phosphotryptic Mapping

Following transfer to Immobilon, the phosphoamino acid content of RPTP was analyzed as described (35) . Phosphopeptide maps of in vivo and in vitroP-labeled proteins were prepared from proteins blotted to nitrocellulose membranes as described (36) . The liberated peptides were dissolved in pH 1.9 buffer, applied to 100-µm thin-layer cellulose plates, and separated by electrophoresis at pH 1.9 for 30 min at 1.5 kV, followed by ascending chromatography in phosphochromotography buffer (37) .

Site-directed Mutagenesis

Oligonucleotides AGGCGGAAAGCGTTGGAATGA (Ser-180 to Ala) and CCTGTTGGTGGCCGGGGACCT (Ser-204 to Ala) were synthesized on a Cyclone DNA synthesizer (Biosearch). Mutagenesis was carried out using the Amersham oligonucleotide-directed in vitro mutagenesis system according to the manufacturer's manual as described (38) . The S180A and S204A single mutations and the double mutation were introduced into pGEXKGPTP and verified by sequencing.

Immunofluorescence Confocal Microscopy

Human 293 cells were trypsinized 24 h following transfection and seeded onto Lab-Tek-chambered glass slides (Nunc) that had been treated with 20 µg/ml poly-L-lysine. The cells were grown in DMEM containing 0.5% CS for 24 h and treated with 100 ng/ml TPA or left untreated for controls. Cells were fixed in freshly made 4% paraformaldehyde/PBS at room temperature for 15 min, rinsed three times for 10 min in 0.5 mM glycine/PBS, permeabilized in 0.5% Triton X-100, 0.5 mM glycine/PBS for 10 min, rinsed three times for 10 min in 0.5 mM glycine/PBS, and blocked in 2% normal goat serum, 0.5 mM glycine/PBS for 20 min. Primary anti-RPTP antibody 5478 was then added to a final dilution of 1:500 and incubated for 1 h in a humidified chamber. The cells were then washed three times for 10 min in 2% normal goat serum, 0.5 mM glycine/PBS, and secondary fluorescein-conjugated goat anti-rabbit Ig (Cappel Labs) was added at a final dilution of 1:50 in 2% normal goat serum, 0.5 mM glycine/PBS and incubated for 20 min. The cells were then washed three times for 10 min in 2% normal goat serum, 0.5 mM glycine/PBS, coverslips were applied to the slides, and the cells were viewed on an Noran Odyssey laser confocal microscope.


RESULTS

RPTP Is a 140-kDa Phosphoprotein Phosphorylated on Serine and Tyrosine Residues, and the Level of Serine Phosphorylation Increases following TPA Stimulation

To study RPTP in vivo, we raised an antiserum against the predicted C-terminal 19 residues of RPTP (Fig. 1 A) and additional antisera against bacterially expressed GST fusion proteins containing either the first catalytic domain or the whole cytoplasmic domain of RPTP. The anti-peptide serum precipitated a 140-kDa protein from [S]methionine/cysteine-labeled NIH3T3 cells (Fig. 1 A). Inclusion of the immunizing peptide specifically blocked precipitation of p140. The anti-bacterial fusion protein sera also precipitated p140 (data not shown). The major [S]methionine/cysteine-labeled tryptic peptides of immunoprecipitated p140 were also present in the 100-kDa product of an in vitro translation reaction of synthetic RPTP mRNA (data not shown), thus demonstrating that p140 is RPTP. The size discrepancy is presumably due to glycosylation of p140; there are 8 predicted N-linked glycosylation sites in the extracellular domain of RPTP. Treatment of NIH3T3 cells with tunicamycin reduced its size from 130 to 110 kDa (data not shown), indicating that RPTP is N-glycosylated. The residual size difference is presumably due to O-glycosylation events (39) . Pulse-chase analysis indicated that RPTP maturation proceeds through a 90-kDa precursor form (data not shown). Daum et al.(39) have reached similar conclusions.


Figure 1: RPTP is a 140-kDa phosphoprotein phosphorylated predominantly on serine residues. A, an antiserum raised against the C-terminal 19 residues of RPTP precipitated a 140-kDa protein from metabolically S-labeled NIH3T3 cells (preimmune, lane1; immune, lane2). Precipitation was blocked by the addition of peptide (immune + peptide, lane3). Proteins were analyzed on a 7.5% SDS-polyacrylamide gel. The gel was impregnated with 2,5-diphenyloxazole and exposed to Kodak XAR film for 6 days at -70 °C. B, immunoprecipitation of RPTP from in vivoP-labeled resting (control, lane1) and TPA-stimulated ( TPA, lane2) NIH3T3 cells. Lanes1 and 2 are nonadjacent lanes from the same gel. Immunoprecipitated RPTP was resolved on a 7.5% SDS-polyacrylamide gel. Proteins were transferred to Immobilon and exposed to Kodak XAR film for 16 h at -70 °C with an intensifier screen. C-F, phosphoamino acid analysis of P-labeled RPTP from resting ( C and E) and TPA-stimulated NIH3T3 cells ( D and F). E and F are 10-day exposures illustrating the presence of phosphothreonine and phosphotyrosine, while C and D are 3-day exposures illustrating the increase in phosphoserine. The horizontalarrowhead indicates the position of phosphotyrosine. The schematic indicates the relative positions of the ninhydrin-stained phosphoamino acid markers.



To determine whether RPTP is phosphorylated, we immunoprecipitated RPTP from P-labeled NIH3T3 cells. RPTP was found to be phosphorylated in cells growth arrested in low serum (Fig. 1 B). Several mitogens were tested to determine whether exogenous stimulation of NIH3T3 cells altered RPTP phosphorylation. The level of phosphorylation increased modestly following stimulation with TPA (Fig. 1 B), platelet-derived growth factor (data not shown), and serum. In different experiments, the level of RPTP phosphorylation increased 30-50% following TPA treatment (Fig. 1 B), and the increase was maximal by 15 min following TPA stimulation (data not shown). Phosphoamino acid analysis of RPTP showed it to be phosphorylated predominantly on serine and to a lesser extent on threonine and tyrosine (Fig. 1, C and E). The increase in phosphorylation detected after TPA treatment was reflected in an increase in phosphoserine (Fig. 1, D and F).

PKC Phosphorylates Bacterially Expressed RPTP in Vitro at the Same Sites as the Major in Vivo Sites (Ser-180 and Ser-204)

To determine if the increase in phosphorylation of RPTP after TPA treatment was due to direct phosphorylation by PKC, the entire cytoplasmic domain of RPTP (residues 168-793) was expressed in bacteria as a GST fusion protein, purified, and phosphorylated in vitro by PKC purified from rat brain (Fig. 2 A). Two-dimensional phosphotryptic mapping of in vivoP-labeled RPTP revealed three major phosphopeptides and several minor ones (Fig. 2 B). The pattern of phosphopeptides was unchanged after TPA treatment, but the intensity of the major phosphopeptides increased. Comparison of this pattern with the phosphotryptic map from in vitro phosphorylated bPTP showed that three of the peptides found in in vivoP-labeled RPTP were generated upon PKC phosphorylation. This was confirmed by the comigration of the in vivo and in vitro generated phosphopeptides (Fig. 2 B).


Figure 2: Bacterially expressed RPTP is phosphorylated in vitro by purified PKC at the same sites as endogenous RPTP. A, the cytoplasmic domain of RPTP was fused to the glutathione S-transferase gene of the expression plasmid pGEXKG (pGEXPTP). The fusion protein was purified on glutathione-Sepharose beads and cleaved with thrombin generating bPTP. The products of an in vitro phosphorylation reaction containing PKC and 0, 5, 25, or 50 ng of bPTP ( lanes1-4) were resolved on a 10% SDS-polyacrylamide gel, transferred to nitrocellulose, and exposed to Kodak XAR film for 15 min. Positions of PKC and bPTP are indicated. Molecular weight standards are Rainbow markers (Amersham). B, phosphotryptic peptide maps of in vivoP-labeled RPTP and cytoplasmic bPTP phosphorylated in vitro by PKC. Approximately 100 cpm were analyzed in each case. Arrows indicate the origin. Electrophoresis was in the horizontal dimension with the cathode on the right. Panel1, control; panel2, TPA-stimulated NIH3T3 cells labeled with [P]orthophosphate as in Fig. 1 B; panel3, in vitro phosphorylated bPTP; panel4, schematic diagram indicating the position of the major phosphopeptides (phosphopeptides found in vivo and in vitro ( A-C) and phosphopeptides found only in vivo (1, 2)); panel5, mix of 50 cpm of control and 50 cpm of in vitro phosphorylated bPTP; panel6, mix of 50 cpm of TPA-stimulated and 50 cpm of in vitro phosphorylated bPTP. Plates were dried and exposed for 6 days at -70 °C with an intensifier screen.



The sequence of RPTP was then examined for tryptic peptides containing possible PKC phosphorylation sites (Ser/Thr-hydrophobic-Arg) (40) . Due to its neighboring basic residues and position near the transmembrane domain, characteristics similar to previously described PKC phosphorylation sites (41) , Ser-180 was chosen as a likely candidate for a PKC site. Ser-180 was mutated to Ala, and the S180A mutant RPTP was expressed in bacteria and phosphorylated in vitro by PKC. The phosphotryptic map of the S180A mutant bPTP revealed the loss of peptides B and C, but peptide A remained (Fig. 3 B), indicating that peptides B and C contain Ser-180. The predicted mobility of the tryptic peptide containing Ser-180 is approximately that of peptide B. The N-terminal residue of this peptide is Gln, and cyclization of this residue to pyrocarboxylic acid during the tryptic mapping procedure would yield a more negatively charged, less hydrophilic species at pH 1.9 corresponding to peptide C; a similar situation exists for the major phosphopeptide derived from the autophosphorylated v-Fps protein (42) . Therefore, we believe that peptide C is derived from peptide B during the generation of the tryptic digest.


Figure 3: Mutation of Ser-180 and Ser-204 abolishes phosphorylation of bPTP by PKC in vitro. A, in vitro phosphorylation of wild type ( WT) and mutant bPTP by purified PKC was performed as in Fig. 2 A. The products of an in vitro phosphorylation reaction containing either no added protein ( lane1), 50 ng of purified GST protein from bacteria expressing the vector alone ( lane2), 50 ng of wild type bPTP ( lane3), 50 ng of S204A bPTP ( lane4), or 50 ng of S180A/S204A bPTP ( lane5) were analyzed on a 10% SDS-polyacrylamide gel, dried, and autoradiographed. The positions of PKC and bPTP are indicated. B, phosphotryptic maps of wild type and the Ser-180 to Ala mutant (S180A) and Ser-204 to Ala mutant (S204A) cytoplasmic domains expressed in bacteria and phosphorylated in vitro by PKC. Panel1, wild type bPTP (2000 cpm); panel2, Ser-180 to Ala mutant (S180A) bPTP (2,000 cpm); panel3, mix of 1000 cpm each of wild type and S180A bPTP; panel4, wild type bPTP (2000 cpm); panel5, Ser-204 to Ala mutant (S204A) bPTP (2000 cpm); panel6, mix of 1000 cpm each of wild type and S204A bPTP. Because the experiment shown in panels1-3 was carried out on a different occasion to that in panels4-6, two wild type controls are shown. Plates were exposed to Kodak XAR film for 16 h at 23 °C. C, schematic showing the position of PKC phosphorylation sites in RPTP. The inset depicts the sequence of the predicted CNBr fragment from bPTP (the upstream methionine is provided by GST) and shows Ser-180 and Ser-204. The sequence begins one residue inside the transmembrane domain as indicated by the horizontalarrow. Arrowheads indicate predicted sites of trypsin cleavage.



To narrow the list of possible tryptic peptides responsible for the remaining peptide A, manual Edman degradation was performed. Free phosphate was released at the third cycle of degradation, indicating that the phosphoserine was the third residue from the N terminus of the peptide (data not shown). Two RPTP peptides met this criterion. The tryptic peptide containing Ser-204 was considered the most likely candidate due to its relatively hydrophilic nature. It was also noted that Ser-204 is located in the same cyanogen bromide (CNBr) fragment as Ser-180. If Ser-204 is the second site of PKC phosphorylation, then in vitro phosphorylation of bPTP should generate a labeled CNBr fragment of 7 kDa, and trypsin digestion of this fragment should yield all three major phosphopeptides present in the intact protein. In addition, the S180A mutant bPTP should generate the same CNBr fragment containing only peptide A. To test this hypothesis, in vitro phosphorylated wild type and S180A bPTP were isolated from an SDS-polyacrylamide gel, excised, cleaved with CNBr, and run on a 20% SDS-polyacrylamide gel. As predicted, a 7-kDa P-labeled band was obtained in both cases. The isolated bands were subjected to tryptic digestion and peptide mapping, and the phosphopeptides were shown to be identical to those obtained following phosphorylation of the intact protein (data not shown).

To confirm the identification, Ser-204 was mutated to alanine, and the S204A mutant bPTP was expressed in bacteria, purified, and phosphorylated in vitro. the S180A/S204A double mutant bPTP failed to be phosphorylated in vitro by PKC (Fig. 3 A). Moreover, the major phosphopeptide, peptide A, was missing from the peptide map of S204A mutant bPTP while peptides B and C were still present (Fig. 3 B). Therefore, we conclude that Ser-204 is the other site of PKC phosphorylation in RPTP. The remaining band in the S180A/S204A lane of approximately the same mobility as bPTP was shown to be identical to a background band in the GST control lane by tryptic peptide mapping (data not shown). Fig. 3 C illustrates the position of the two phosphorylation sites relative to the membrane spanning domain and the two catalytic domains of RPTP.

Transient Expression of Wild Type and S180A/S204A RPTP in 293 Cells

To examine the effect of the phosphorylation site mutations in vivo, sequences encoding either the entire wild type or the S180A/S204A RPTP were expressed from the Sg5 vector by transient transfection of 293 cells, a human embryonic kidney cell line immortalized with adenovirus E1A (Fig. 4 A). As determined by immunoprecipitation of S-labeled cells, the level of RPTP expression in transfected cells was greater than 20 times that in the vector-transfected controls (Fig. 4 A). The protein migrating at 87 kDa in transfected cells was shown to have an identical [S]methionine/cysteine-labeled tryptic map to RPTP (data not shown) and is likely to be the unglycosylated RPTP precursor (39) .() Treatment of P-labeled wild type RPTP-expressing 293 cells with TPA resulted in a large increase in phosphorylation of RPTP (Fig. 4 B). In contrast, TPA treatment of cells expressing the S180A/S204A mutant RPTP resulted in only a very slight increase in P incorporation. Tryptic peptide mapping revealed that in resting cells, wild type RPTP was phosphorylated on the same three major peptides that were phosphorylated in vitro by PKC (data not shown). Peptide maps of S180A/S204A mutant RPTP in resting cells contained a single phosphopeptide that comigrated with the phosphopeptide that contains Ser-204. This peptide contained only phosphoserine (data not shown). TPA treatment of S180A/S204A RPTP-expressing cells did not increase the P content of this major peptide but rather at a large number of sites as demonstrated by the presence of a number of new phosphopeptides. Since PKC did not phosphorylate the S180A/S204A mutant RPTP in vitro (Fig. 3 B), 293 cells may contain a protein kinase distinct from PKC that phosphorylates RPTP at Ser-202, which is present in the same tryptic peptide as Ser-204.


Figure 4: Transient expression of wild type and S180A/S204A mutant RPTP in human embryonic kidney (293) cells. 293 cells were transiently transfected overnight with the Sg5 expression vector or Sg5 encoding either full-length wild type ( WT) or S180A/S204A RPTP. The following morning, the medium was changed from DMEM containing 10% calf serum to DMEM containing 1% serum. After 8 h, the cells were metabolically labeled with either [S]methionine/cysteine ( A) or [P]orthophosphate ( B), and after an additional 16 h, the cells were either treated with 100 ng/ml TPA for 10 min(+) or left untreated (-). RPTP was then immunoprecipitated with anti C-terminal peptide antiserum and analyzed on a 7.5% SDS-polyacrylamide gel.



Down-regulation of PKC Results in Reduced TPA-stimulated Phosphorylation of RPTP

It has been well documented that long term exposure to TPA leads to down-regulation of some isoforms of PKC. Reduced phosphorylation of RPTP following preincubation with TPA would be taken as evidence that PKC was in fact the protein kinase responsible for the in vivo phosphorylation. To test this experimentally, 293 cells were transiently transfected with the expression vectors for wild type RPTP or the S180A/S204A mutant RPTP, serum-starved, and labeled with [S]methionine/cysteine or [P]orthophosphate, and, where indicated, cells were pretreated with 100 ng/ml TPA for 24 h to down-regulate PKC (Fig. 5). In cells expressing wild type RPTP, the increase in phosphorylation seen following TPA stimulation was reduced when the cells were preincubated with TPA. TPA-stimulated phosphorylation was also reduced in cells expressing the S180A/S204A mutant RPTP, indicating that the sites that are phosphorylated in the absence of the major PKC sites are also due to PKC activation and presumably result from phosphorylation by a PKC-activated protein kinase. It is interesting to note that there was a small (50%) but reproducible increase in the apparent amount of S-labeled RPTP present in immunoprecipitates from RPTP-expressing transfected cells that had been treated with TPA (Fig. 5). The increase was also apparent in immunoblots of total cell lysates and in RPTP immunoprecipitates from cells treated with TPA in the presence of cycloheximide (data not shown). This result indicates that the apparent increase in RPTP was not the result of new protein synthesis, which was to be expected given the short (10 min) TPA treatment. The reported values for the increase in phosphorylation have been calculated, taking the increase in the apparent amount of RPTP protein into account.


Figure 5: Down-regulation of PKC results in reduced TPA-stimulated phosphorylation of RPTP. Human 293 cells were transiently transfected, serum-starved, in vivo labeled, immunoprecipitated, and analyzed as in Fig. 4 except that, where indicated, cells were pretreated with 100 ng/ml TPA for the 24 h that the cells were incubated in low serum. Upper left, P-labeled wild type-transfected 293 cells. Upper right, P-labeled S180A/S204A-transfected 293 cells. Lower left, S-labeled wild type-transfected 293 cells. Lower right, S-labeled S180A/S204A-transfected 293 cells.



Immunofluorescence Staining of 293 Cells Transiently Expressing RPTP

The puzzling observation that the amount of RPTP protein apparently increased upon TPA treatment prompted us to examine the subcellular location of RPTP in transfected 293 cells using immunofluorescence confocal microscopy. RPTP-expressing cells could readily be detected by this method and represented 30-50% of the total population, depending on the experiment (Fig. 6). The subcellular distributions of both wild type and S180A/S204A mutant RPTP were identical (data not shown). In the majority of the transfected cells, RPTP was present at the cell surface as expected (Fig. 6, D-H). However, in 30-40% of the positive cells, RPTP staining appeared in dense intracellular granules that were clearly distinct from the Golgi apparatus (Fig. 6 B). Stimulation cells with TPA resulted in a transient apparent increase in the percentage of cells expressing exogenous RPTP at the cell surface (Fig. 6 D). The TPA-induced redistribution of RPTP may reflect not only translocation but also maturation of the protein into the 140-kDa form, thus accounting for the apparent protein synthesis-independent increase in RPTP in TPA-treated cells.


Figure 6: Immunofluorescence staining of RPTP in transiently transfected 293 cells. 293 cells were trypsinized 24 h following transfection with Sg5 wild type RPTP expressing and seeded on to chambered glass slides. The cells were grown in DMEM containing 0.5% serum for 24 h and treated with 100 ng/ml TPA for 10 min or left untreated for control. The cells were stained with affinity-purified antibodies raised against bPTP (5478). A, phase contrast of a field of control cells. B, the same field of cells viewed with fluorescence. A positive staining cell is in the center of the field. Almost all of the stain is localized within intracellular granules; very little or none was visible at the cell surface. C, phase contrast of a field of TPA-treated cells. D-E, the same field of cells viewed with fluorescence and focused in D at the level of the substratum and in E on the top of the cell. The majority of the staining can be seen at the cell surface. F-H, other TPA-treated cells exhibiting either entirely surface-associated staining ( F and G) or localized to both the cell surface and intracellular granules ( H).




DISCUSSION

In this study, we have shown that RPTP is a phosphoprotein. In fibroblasts, RPTP is phosphorylated on serine at two major sites, Ser-180 and Ser-202, and as we have reported elsewhere (25) , on Tyr-789. The phosphorylation of Ser-180 and Ser-202 is stimulated by TPA treatment, and, since these sites are phosphorylated in vitro by PKC, we conclude that they are phosphorylated by PKC in vivo. In NIH3T3 cells, the stimulation by TPA was only modest but much more dramatic when exogenous RPTP was expressed in 293 cells. RPTP is not an abundant protein in fibroblasts, and we suspect that basal PKC activity is sufficient to phosphorylate Ser-180 and Ser-204 even under growth-arrested conditions. We have not determined the exact stoichiometry of phosphorylation at these sites, but based on our estimate of 1.2 mol of phosphate per mol of RPTP in NIH3T3 cells (25) , and the relative intensities of the Ser-180/Ser-202 peptides and the Tyr-789 peptide, the stoichiometry of phosphorylation of Ser-180 and Ser-202 is probably close to 50% in NIH3T3 cells. Thus, a 2-fold stimulation of phosphorylation is the maximum that can be achieved. In 293 cells, where exogenous RPTP is overexpressed 20-fold, the basal level of phosphorylation is much lower, and PKC-dependent phosphorylation is much more dramatic.

Our results indicate that Ser-180 and Ser-204 are directly phosphorylated by PKC in vivo. Ser-180 is in a conventional PKC consensus sequence, and this, coupled with its location 14 residues from the inner face of the plasma membrane, makes it an ideal target for PKC following its translocation to the plasma membrane in response to increased levels of diacylglycerol. Ser-204, on the other hand, is not in a perfect PKC consensus sequence because the Arg on the C-terminal side is at the +3 position rather than at +2 (40) . However, the Arg at -3 on the N-terminal side of Ser-204, coupled with its location close to the plasma membrane, may compensate for this. Although RPTP and RPTP are closely related proteins, RPTP lacks a Ser at the position equivalent to Ser-180. RPTP does have a Ser at the position equivalent to Ser-204, and although the surrounding sequence is not identical, the two basic residues are conserved, and this Ser could be a substrate for PKC. Ser-202, which may be phosphorylated in the S204A mutant RPTP, is followed by a proline and could be phosphorylated by a proline-directed protein kinase such as a MAP kinase. We note that several other RPTPs, including LAR, RPTP, and RPTP, have potential PKC phosphorylation sites within 20 residues of the inner face of the plasma membrane.

There are many precedents for membrane-associated proteins that are phosphorylated and regulated by PKC. For instance, the EGF receptor is phosphorylated by PKC at Thr-654, which is in the juxtamembrane domain in an exactly analogous location to Ser-180/Ser-204 (41) , and this reduces EGF-dependent activation of the EGF receptor protein-tyrosine kinase (43) . Another membrane protein, CD4, has been shown to be phosphorylated in a similar location in response to TPA. The phosphorylation of CD4 by PKC also appears to be functionally important (44, 45) .

Does PKC phosphorylation of RPTP in the juxtamembrane domain regulate its activity? den Hertog and colleagues (19) have shown that TPA stimulation of 293 or P19 cells expressing exogenous RPTP increases the in vitro PTP activity of RPTP 2-3-fold, due to a decreased Kfor phosphorylated myelin basic protein substrate rather than an increase in V. The increased activity is reduced by phosphatase treatment of RPTP, probably as a result of dephosphorylation of Ser-180/Ser-204, which are phosphorylated in response to TPA in these cells but possibly of other sites as well. To determine whether phosphorylation Ser-180/Ser-204 directly affects RPTP activity, we phosphorylated bPTP with purified PKC. Phosphorylation did not affect its activity in vitro (data not shown), but the maximal phosphorylation stoichiometry we could achieve was 10%, which would have prevented us from detecting a small change in bPTP activity. We have also been unable to find a conclusive difference in activity between wild type and S180A/S204A mutant RPTP expressed in 293 cells upon TPA treatment (data not shown), but this analysis is complicated by the TPA-induced increase in the mature form of RPTP (Fig. 5). It is also possible that phosphorylation at other sites affects RPTP activity. At present, it is an open question whether a small decrease in Kwill significantly affect RPTP function in vivo, but if RPTP activity is stimulated via PKC-mediated phosphorylation in vivo, then this could provide a mechanism for down-regulating the activity of ligand-activated receptor-like protein-tyrosine kinases, such as the platelet-derived growth factor receptor, that stimulate phosphoinositide turnover, either by dephosphorylating the receptor itself or substrates phosphorylated by the receptor.

Two other PTPs are known to be phosphorylated by PKC. In vitro PKC phosphorylates PTP1B at Ser-378, and phosphorylation of Ser-378 is stimulated in TPA-treated cells (17) . However, phosphorylation of Ser-378 does not appear to affect PTP1B activity. PTP-PEST is phosphorylated in vitro by PKC and the cAMP-dependent protein kinase at Ser-39 and Ser-435, and the same sites are phosphorylated in TPA- or forskolin-treated HeLa cells (20) . In this case, phosphorylation at Ser39 decreases PTP-PEST activity by increasing its Kfor substrate.

Many PTPs are discretely localized in the cell (46) , and the subcellular localization of PTPs presumably dictates access to substrate. Given its primary structure and the fact that it is a heavily glycosylated protein, one might expect RPTP to be found on the cell surface. We attempted to determine the subcellular distribution of RPTP by immunofluorescence staining, but we were unable to obtain convincing staining of endogenous RPTP in either NIH3T3 cells or in untransfected 293 cells due to the low level of protein. Therefore, we resorted to the use of transiently transfected 293 cells to localize RPTP, but it should be kept in mind that RPTP is highly overexpressed in this system, and this may affect its subcellular localization. Nevertheless, with this reservation we found that most cells displayed surface staining as expected if RPTP is localized to the plasma membrane. However, as assessed by immunofluorescence confocal microscopy, the subcellular distribution of RPTP varied from entirely intracellular to entirely at the cell surface. The nature of the dense intracellular granules that stained in 30-40% of the RPTP positive cells is unknown, but they were clearly distinct from the Golgi apparatus. TPA stimulation resulted in a transient increase in the fraction of cells expressing exogenous RPTP at the cell surface. This translocation did not require direct phosphorylation of RPTP by PKC because the S180A/S204A double mutant RPTP behaved in a similar fashion (data not shown). TPA also rapidly increased the amount of the 140-kDa form of RPTP in transiently transfected 293 cells, suggesting that a PKC-mediated process accelerates the maturation of RPTP. This effect of TPA was also detected in NIH3T3 cells, suggesting that it is a normal cellular response. The relocalization of RPTP may modulate its access to specific substrates. Thus, activation of PKC by cytokines can in principle affect the amount of mature RPTP, its localization, and activity, and all of these may regulate RPTP function.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants CA14195 and CA39780. 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.

§
Supported by a United States Public Health Service Postdoctoral Training Grant.

Present address: Mount Sinai Hospital Research Institute, 600 University Ave., Toronto, Ontario M5G 1X5, Canada.

**
An American Cancer Society Research Professor. To whom correspondence should be addressed.

The abbreviations used are: PTP, protein-tyrosine phosphatase; TPA, 12- O-tetradecanoylphorbol-13-acetate; PKC, protein kinase C; DMEM, Dulbecco's modified Eagle's medium; PBS, phosphate-buffered saline; DTT, dithiothreitol; RPTP, receptor-like protein-tyrosine phosphatase; MAP, mitogen-activated protein; EGF, epidermal growth factor; GST, glutathione S-transferase; bPTP, bacterial protein-tyrosine phosphatase ; CS, calf serum.

J. den Hertog, personal communication.


ACKNOWLEDGEMENTS

We are grateful to Ushio Kikkawa for supplying purified protein kinase C, to Carl Hoeger and Jean Rivier for synthesis of the C-terminal RPTP peptide, and to Jeroen den Hertog for communicating unpublished results and providing affinity-purified anti-bPTP antibodies. We thank Dan Chin for carrying out the confocal microscopy.


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