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Originally published In Press as doi:10.1074/jbc.M507722200 on October 31, 2005

J. Biol. Chem., Vol. 280, Issue 52, 42694-42700, December 30, 2005
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Receptor-type Protein-tyrosine Phosphatase-{kappa} Regulates Epidermal Growth Factor Receptor Function*

Yiru Xu, Li-Jun Tan, Vladimir Grachtchouk, John J. Voorhees, and Gary J. Fisher1

From the Department of Dermatology, University of Michigan, Ann Arbor, Michigan 48109

Received for publication, July 15, 2005 , and in revised form, October 28, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Epidermal growth factor receptor (EGFR), the prototypic receptor protein tyrosine kinase, is a major regulator of growth and survival for many epithelial cell types. We report here that receptor-type protein-tyrosine phosphatase-{kappa} (RPTP-{kappa}) dephosphorylates EGFR and thereby regulates its function in human keratinocytes. Protein-tyrosine phosphatase (PTP) inhibitors induced EGFR tyrosine phosphorylation in intact primary human keratinocytes and cell-free membrane preparations. Five highly expressed RPTPs (RPTP-beta, {delta}, {kappa}, µ, and {xi}) were functionally analyzed in a Chinese hamster ovary (CHO) cell-based expression system. Full-length human EGFR expressed in CHO cells, which lack endogenous EGFR, displayed high basal (i.e. in the absence of ligand) tyrosine phosphorylation. Co-expression of RPTP-{kappa}, but not other RPTPs, specifically reduced basal EGFR tyrosine phosphorylation. RPTP-{kappa} also reduced epidermal growth factor-dependent EGFR tyrosine phosphorylation in CHO cells. Purified RPTP-{kappa} preferentially dephosphorylated EGFR tyrosines 1068 and 1173 in vitro. Overexpression of wild-type or catalytically inactive RPTP-{kappa} reduced or enhanced, respectively, basal and EGF-induced EGFR tyrosine phosphorylation in human keratinocytes. Furthermore, siRNA-mediated knockdown of RPTP-{kappa} increased basal and EGF-stimulated EGFR tyrosine phosphorylation and augmented downstream Erk activation in human keratinocytes. RPTP-{kappa} levels increased in keratinocytes as cells reached confluency, and overexpression of RPTP-{kappa} in subconfluent keratinocytes reduced keratinocyte proliferation. Taken together, the above data indicate that RPTP-{kappa} is a key regulator of EGFR tyrosine phosphorylation and function in human keratinocytes.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein tyrosine phosphorylation is a reversible post-translational modification that modulates a variety of protein functions, including protein stability, protein-protein interaction, enzymatic activity, etc. Regulated reversible protein phosphorylation plays key roles in signal transduction pathways controlling many fundamental cellular processes, such as proliferation, differentiation, motility, metabolism, cytoskeletal organization, development, and cell-cell interactions (1, 2). The level of protein tyrosine phosphorylation is controlled by two classes of counteracting enzymes, namely protein-tyrosine kinases (PTKs2, EC 2.7.1.112 [EC] ) and protein-tyrosine-phosphatases (PTPs, EC 3.1.3.48 [EC] ). The number of genes in the human genome that encode active PTPs and active PTKs has recently been estimated to be very similar (3, 4). Emerging evidence indicates that, depending on the particular pathway, protein tyrosine dephosphorylation can be of equal or greater importance than protein tyrosine phosphorylation for the regulation of cellular function (5).

Epidermal growth factor receptor (EGFR, ErbB1) belongs to the receptor protein-tyrosine kinase (RPTK) superfamily. It is composed of an extracellular ligand binding domain, a single transmembrane domain, and an intracellular domain possessing PTK activity. Ligand binding to the extracellular domain of EGFR stabilizes homodimerization and heterodimerization with other ErbB members, which promotes trans tyrosine phosphorylation of the intracellular C-terminal domain. EGFR activation is synonymous with increased phosphorylation of specific tyrosine residues within its intracellular C-terminal domain. These phosphorylated tyrosines function as docking sites for a variety of signaling molecules that regulate membrane-proximal steps of signal transduction cascades that ultimately bring about cellular responses to EGFR ligands (6). Recent data suggest that EGFR not only participates in cognate ligand-induced signal transduction pathways but also plays important roles in diverse signal transduction pathways initiated by G protein-coupled receptors, cytokine receptors, integrins, ion channels, and stress responses (7–9).

Aberrant regulation of EGFR has been shown to promote multiple tumorigenic processes by stimulating proliferation, angiogenesis, and metastasis (10). EGFR and/or its ligands have a critical role in most common human epithelial cancers and many different types of solid tumors (11). The central role of EGFR in diverse signal transduction pathways dictates that its tyrosine phosphorylation must be strictly regulated. One potential mechanism for such regulation is through PTP-catalyzed dephosphorylation.

The subfamily of "classical," strictly tyrosine-specific PTPs contains 38 members, 21 of which are transmembrane receptor types and 17 of which are intracellular, non-receptor types (4). All classical PTPs contain a signature motif (HVCXXXXXR(S/T)) within a catalytic domain of 250 amino acid residues (12). The cysteine residue in the PTP signature motif is absolutely required for catalytic activity (13). The receptor-type PTPs (RPTPs) are integral membrane proteins composed of extracellular adhesion molecule-like domains, a single transmembrane domain, and a cytoplasmic domain containing one or two catalytic domains.

Reduced phosphorylation of EGFR has been associated with several different PTP activities (14–18). However, identification of RPTP(s) that directly dephosphorylate(s), and thereby regulate(s), EGFR function is lacking. Using an expression strategy in EGFR-lacking Chinese hamster ovary (CHO) cells, we have identified RPTP-{kappa} as a specific EGFR PTP. We have further demonstrated that RPTP-{kappa} regulates both basal and ligand-induced EGFR tyrosine phosphorylation and function.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Adult human primary keratinocytes were obtained from Cascade Biologics Inc. (Portland, OR). CHO cells were purchased from American Type Culture Collection (Manassas, VA). EGFR, Erk, and RPTP-beta antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). PD169540 was generously provided by Dr. David Fry from Pfizer (Ann Arbor, MI). Phospho-EGFR (pY-1068 and pY-992) and phospho-Erk antibodies were purchased from Cell Signaling Technology (Beverly, MA). Phospho-EGFR (pY-1173) was purchased from Upstate Biotechnologies (Lake Placid, NY). RPTP-µ antibody was purchased from Exalpha Biologicals, Inc. (Watertown, MA).

Cell Culture—Adult human primary keratinocytes were expanded in modified MCDB153 medium (EpiLife, Cascade Biologics, Inc.). CHO cells were cultured in Ham's F-12 medium with 1.5 g/liter sodium bicarbonate supplemented with 10% fetal bovine serum.

Preparation of Keratinocyte Membranes and EGFR Activation Assay— Human keratinocytes were washed twice with ice-cold hypotonic buffer (20 mM Tris-HCl, pH 7.6 with 10 mM NaCl) supplemented with 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, and 1 mM phenylmethylsulfonyl fluoride. Cells were disrupted in a Dounce homogenizer. Lysates were centrifuged at 500 x g for 10 min, and the supernatant was centrifuged at 20,000 x g for 30 min. Membranes were extracted with 0.5 M NaCl to remove loosely associated proteins. Membrane suspension was then supplemented with 100 µM ATP, 0.2% beta-mercaptoethanol and 30 mM MgCl2. Incubation with EGF or phosphatase inhibitors was performed at room temperature. SDS sample buffer was added to stop the reactions. EGFR tyrosine phosphorylation was analyzed by Western blot using phospho-EGFR (pY-1068) antibody.

Preparation of Whole Cell Lysates—Human primary keratinocytes or CHO cells were washed twice with ice-cold phosphate-buffered saline, scraped from the culture plates in WCE buffer (25 mM Hepes, pH 7.2, 75 mM NaCl, 2.5 mM MgCl2, 0.2 mM EDTA, 0.1% Triton X-100, 0.5 mM dithiothreitol, 20 mM beta-glycerophosphate) supplemented with 10 µg/ml aprotinin, 10 µg/ml leupeptin, 10 µg/ml pepstatin A, 1 mM phenylmethylsulfonyl fluoride, and 1 mM sodium orthovanadate. Following 10 min of incubation at 4 °C, cell homogenates were centrifuged at 14,000 x g for 10 min, and supernatants were collected and used as whole cell lysates.

Protein Phosphatase Assay—Phosphatase assay buffer (100 µl) containing 5 mg/ml p-nitrophenyl phosphate, 100 g/ml bovine serum albumin, 50 mM Tris, pH 7.6, 100 mM NaCl, and 10 mM EDTA was mixed with cell membrane extracts (50 µl) and incubated at 37 °C for 30 min in 96-well microtiter plates. Reactions were terminated by the addition of 13% K2HPO4 (15 µl). Hydrolysis of p-nitrophenyl phosphate was measured spectrophotometrically (405 nm) using a microtiter plate reader (Titertek Multiskan MCC/340, EFLAB, Finland).

Generation of RPTP-{kappa} Polyclonal Antibody—A peptide with a unique sequence derived from the intracellular domain (RGHNESKADCLD-MDP KAPQH) with predicted high probability of surface exposure and high antigenic index was synthesized, conjugated to keyhole limpet hemocyanin, and injected into New Zealand White rabbits (Bethyl Laboratories, Inc., Montgomery, TX). After two booster injections, anti-RPTP-{kappa} antibody was affinity-purified from hyperimmune serum. The antibody was tested for its performance in enzyme-linked immunosorbent assay, Western blot, and immunoprecipitation.

In Vitro Dephosphorylation of Purified EGFR—Purified full-length EGFR was purchased from BIOMOL (Plymouth Meeting, PA). EGFR was tyrosine-phosphorylated according to the manufacturer's protocol and used as substrate for the RPTP-{kappa} intracellular region GST fusion protein. Dephosphorylation reactions were terminated by the addition of SDS sample loading buffer, and the level of EGFR tyrosine phosphorylation was measured by Western analysis probed with phospho-EGFR antibody.

Western Analysis Detection and Quantitation—Western blots were developed and quantified using an enhanced chemifluorescence detection system (Amersham Biosciences). Immunoreactive fluorescent protein bands were detected by the STORM phosphor-imaging device using ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Sample loads, antibody concentration, and incubation times were adjusted to yield fluorescent signals within the linear range of detection.

Transient Transfection of CHO Cells—Mammalian expression vectors for EGFR (pRK5 EGFR) and RPTP (pShuttle RPTP) coding sequences were transiently transfected into CHO cells using Lipofectamine 2000 (Invitrogen). Expression of EGFR and RPTP mRNA and protein was confirmed by real-time reverse transcription (RT)-PCR (7700 Taqman Sequence Detector, Applied Biosystems, Foster City, CA) and Western analysis, respectively, 24 h after transfection.

Association of EGFR with Wild-type and Trapping Mutant RPTP-{kappa}— Full-length EGFR and His6-tagged full-length wild-type or trapping mutant (D1051A) RPTP-{kappa} were co-transfected into CHO cells. One day after transfection, the cells were treated with 50 ng/ml EGF for 10 min at 37 °C and subsequently lysed in TGH buffer (50 mM Hepes, pH 7.2, 20 mM NaCl, 10% glycerol, and 1% Triton X-100), and RPTP-{kappa} protein complexes were purified using Dynabeads TALON (Dynal Biotech, Oslo, Norway) and analyzed by Western blot.

Adeno-X Expression Vector Construction and Adenovirus Production— Wild-type and catalytically inactive (2C/S) human RPTP-{kappa} expression vectors were generated using the Adeno-X expression system (Clontech Laboratories, Inc., Palo Alto, CA). To facilitate detection of expressed RPTP-{kappa}, a polyhistidine (His6) tag was inserted into the C terminus of RPTP-{kappa}. HEK293 cells were used for adenovirus production and purification.

Generation of RPTP-{kappa}-GST Fusion Protein—cDNA encoding the intracellular region of human RPTP-{kappa} (RPTP-{kappa}-IC, corresponding to residues 754–1414) was cloned into the XhoI (5') and NotI (3') site of GST fusion protein expression vector pGEX-6P-3. GST-RPTP-{kappa}-IC fusion protein was expressed in Escherichia coli strain BL21 and purified by GST affinity column according to the manufacturer's protocol (Amersham Biosciences). Purity was at least 90%, as judged by SDS-PAGE.

siRNA Silencing of RPTP-{kappa} in Primary Human Keratinocytes—A unique 21-mer RNA sequence derived from RPTP-{kappa} coding sequence (5'-AAG GTT TGC CGC TTC CTT CAG-3') was designed using Oligoengine software (Seattle, WA). Control siRNA contained a random sequence without homology to any known human gene. Double-stranded siRNA was synthesized by Xeragon Inc, (Valencia, CA). The synthetic siRNA was transfected into primary human keratinocytes using Amaxa Biosystems Nucleofactor (Cologne, Germany). Expression of RPTP-{kappa} in primary keratinocytes increased with cell confluency (see Fig. 10A). To achieve the maximal knockdown effect, siRNA-transfected keratinocytes were cultured to confluency for analysis.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Inhibition of Protein-Tyrosine-Phosphatase Activity Is Associated with Increased EGFR Tyrosine Phosphorylation in Primary Human Keratinocytes—Treatment of intact primary adult human keratinocytes with two nonspecific PTP inhibitors, H2O2 and pervanadate, caused substantial and rapid tyrosine phosphorylation of EGFR (Fig. 1A). The magnitude of EGFR tyrosine phosphorylation was similar to that induced by EGF (20 ng/ml) (Fig. 1A). For these, an antibody that specifically recognizes phosphorylated tyrosine 1068 in EGFR was used. This approach allowed us to detect phosphorylated EGFR in lysates without the need for immunoprecipitation prior to Western blotting. The finding that PTP inhibitors cause accumulation of EGFR tyrosine phosphorylation suggests the potential importance of PTP activity in the regulation of EGFR function.


Figure 1
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  		FIGURE 1.
Protein-tyrosine-phosphatase inhibitors increase EGFR tyrosine phosphorylation in keratinocytes and isolated keratinocyte membranes. Primary human keratinocytes (A) or isolated membranes (B and C) were treated with vehicle (CTRL), EGF (20 ng/ml), hydrogen peroxide (10 mM, H2O2), pervanadate (1 mM, VH), or orthovanadate (1 mM, VO4) for 10 min. EGFR phosphotyrosine 1068 (pY-1068) and total EGFR (A and B, respectively) were analyzed by Western blot. Results are means ± S.E. of fluorescent band intensities quantified by STORM as described under"Experimental Procedures." Insets show representative Western blots. C, phosphatase activity in isolated membranes treated as described above. n = 3; *, p < 0.05 versus controls (CTRL).

 


Figure 2
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  		FIGURE 2.
Co-expression of RPTP-{kappa} with EGFR in CHO cells. A, CHO cells were transfected with empty or EGFR expression vector and treated 24 h later with vehicle or EGF (20 ng/ml) at 37 °C for 10 min. Whole cell lysates were analyzed by Western blot using antibodies to EGFR phosphotyrosine 1068 (pY-1068) and total EGFR. Results are representative of three experiments. B, CHO cells were co-transfected with human EGFR and the indicated RPTP expression vectors. Total RNA was isolated, and the level of each RPTP transcript was quantified by real-time RT-PCR. None of the RPTPs were detected in the empty vector control cells. C, CHO cells were co-transfected with human EGFR and empty or the indicated RPTP expression vectors. Cell lysates were analyzed by Western blot. Results are representative of three experiments.

 
This potential importance was further investigated in cell-free, EGFR-enriched membrane fractions from human keratinocytes. Treatment of membranes in the presence of Mg2+/ATP with PTP inhibitors hydrogen peroxide, pervanadate, or orthovanadate significantly induced phosphorylation of EGFR tyrosine residue 1068 (Fig. 1B). The magnitude of tyrosine 1068 phosphorylation following treatment with either PTP inhibitor was 3–5-fold greater than treatment of membranes with EGF (20 ng/ml). As expected, treatment of keratinocyte membranes with hydrogen peroxide, pervanadate, or orthovanadate (but not EGF) inhibited endogenous membrane-associated PTP activity (Fig. 1C). These data suggest the possible involvement of an integral membrane RPTP activity in the regulation of EGFR tyrosine phosphorylation.

Profile of RPTPs in EGFR-expressing Human Keratinocytes—To investigate the above possibility, we first assessed which of 21 known RPTPs in the human genome are expressed in human keratinocytes. Specific PCR primers for each of the 21 human RPTPs were designed and tested for specificity using cloned cDNAs as templates. Each of the 21 PCR products generated from cloned templates was authenticated by DNA sequencing. RT-PCR was used to detect mRNA expression of each of the 21 RPTPs in adult human keratinocytes and adult human skin. RT-PCR reactions for 13 RPTPs yielded products of the expected size (data not shown). Each product was cloned and verified by DNA sequencing. RPTPs beta, {delta}, {kappa}, µ, and {zeta} were predominantly expressed and therefore chosen for further study.

Dephosphorylation of EGFR by RPTP-{kappa} Transiently Expressed in CHO Cells—To determine whether any of the five candidate RPTPs is able to regulate EGFR tyrosine phosphorylation, we employed a transient transfection system using CHO cells, which do not express EGFR. Transient transfection of CHO cells with EGFR expression vector alone resulted in a high level of basal (i.e. in the absence of ligand) EGFR tyrosine phosphorylation (Fig. 2A). Treatment of EGFR-expressing CHO cells with EGF modestly increased tyrosine phosphorylation of EGFR (Fig. 2A). Tyrosine phosphorylation of EGFR in CHO cells was completely blocked by treatment of cells with EGFR tyrosine kinase inhibitor PD169540, demonstrating that tyrosine phosphorylation of EGFR in CHO cells was due to intrinsic EGFR tyrosine kinase activity (data not shown).

To examine the ability of the RPTPs to dephosphorylate EGFR, cDNAs encoding human RPTP-beta,-{delta},-{kappa},-µ, and -{zeta} were co-expressed with EGFR in CHO cells. To verify that each RPTP was expressed, we determined their mRNA levels using real-time RT-PCR. In vector control-transfected CHO cells, no mRNA for any of the five RPTPs was detectable. In RPTP-transfected CHO cells, the mRNA level of each of the five RPTPs was readily detectable and similar (Fig. 2B). We also determined that transfection resulted in detectable RPTP-beta,-{kappa}, and -µ protein expression (we could not obtain useful antibodies for RPTP-{delta} and -{zeta}) (Fig. 2C). Importantly, only RPTP-{kappa} (but not RPTP-beta,-{delta},-{kappa},-µ, and -{zeta}) was able to significantly reduce constitutive tyrosine phosphorylation of EGFR in CHO cells (Fig. 3A). In addition to the reduction of constitutive EGFR tyrosine phosphorylation, expression of RPTP-{kappa} reduced EGF-stimulated EGFR tyrosine phosphorylation in CHO cells (Fig. 3B). These results indicate that RPTP-{kappa} is capable of reducing EGFR intrinsic tyrosine kinase-catalyzed phosphorylation when co-expressed in CHO cells.

RPTP-{kappa} Directly Dephosphorylates EGFR in Vitro—To determine whether RPTP-{kappa} can directly dephosphorylate EGFR, we constructed, expressed, and purified catalytically active human RPTP-{kappa} intracellular region GST fusion protein (GST-RPTP-{kappa}-IC). GST-RPTP-{kappa}-IC was incubated with autophosphorylated purified full-length human EGFR, and the rate of tyrosine dephosphorylation was monitored by Western analysis using antibodies specific for phosphotyrosine residues 1068, 992, and 1173. RPTP-{kappa} rapidly dephosphorylated EGFR tyrosine 1068 and 1173 (Fig. 4). EGFR tyrosine 992 was dephosphorylated at a substantially slower rate than tyrosine 1068 (Fig. 4). In subsequent experiments, tyrosine phosphorylation at residue 1068 was chosen to monitor EGFR dephosphorylation by RPTP-{kappa}.


Figure 3
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  		FIGURE 3.
Co-expression of RPTP-{kappa} with EGFR reduces EGFR tyrosine phosphorylation in CHO cells. A, CHO cells were co-transfected with EGFR and the indicated RPTP expression vectors. Whole cell lysates were prepared and analyzed for EGFR phosphotyrosine 1068 (pY-1068) and total EGFR by Western blot. Inset shows representative Western blots. Results are means ± S.E. of fluorescent band intensities quantified by STORM as described under"Experimental Procedures." n = 5; *, p < 0.05 versus empty vector. B, CHO cells were co-transfected with EGFR and either empty or RPTP-{kappa} expression vectors. Cells were treated with vehicle (Veh) or EGF (10 ng/ml) for 5 min at 37 °C. Whole cell lysates were prepared and analyzed for EGFR phosphotyrosine 1068 (pY-1068), total EGFR, and RPTP-{kappa} by Western blot. Results are means ± S.E. of fluorescent band intensities quantified by STORM as described under"Experimental Procedures." n = 4; *, p < 0.05 versus empty vector. Inset shows representative Western blot. *, p < 0.05 versus 0 min.

 


Figure 4
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  		FIGURE 4.
RPTP-{kappa} preferentially dephosphorylates EGFR tyrosine 1068 and 1173 in vitro. Autophosphorylated EGFR was incubated with purified RPTP-{kappa} or GST at room temperature. At the indicated times, reactions were analyzed for EGFR tyrosine phosphorylation at residues 992, 1173, or 1068 by Western blot. Results are means ± S.E. of fluorescent band intensities quantified by STORM as described under"Experimental Procedures." n = 3.

 


Figure 5
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  		FIGURE 5.
EGFR associates with RPTP-{kappa}-trapping mutant in CHO cells. CHO cells were co-transfected with EGFR and empty vector, wild-type His-RPTP-{kappa} (WT), or trapping mutant His-RPTP-{kappa} (DA). One day after transfection, His-tagged RPTP-{kappa} protein complexes in whole cell lysates were purified by nickel-coated beads (Pull-Down). RPTP-{kappa} (WT and DA) and total EGFR were analyzed by Western blot. Fluorescent band intensities were quantified by STORM as described under"Experimental Procedures." n = 2.

 
Association of Substrate-trapping Mutant RPTP-{kappa} and EGFR in Intact Cells—To further investigate dephosphorylation of EGFR by RPTP-{kappa}, we performed substrate-trapping studies. Mutation of an aspartic acid (Asp-1051 for RPTP-{kappa}), which is conserved in the active site of protein-tyrosine phosphatases, to alanine prevents completion of phosphate ester hydrolysis and therefore causes the formation of a stable enzyme-substrate complex (14). We co-expressed full-length human EGFR with His-tagged full-length wild-type or D1051A mutant RPTP-{kappa} in CHO cells. Following expression, RPTP-{kappa} was captured on nickel-coated beads and analyzed for its association with EGFR by Western analysis. Although wild-type RPTP-{kappa} catalyzes the dephosphorylation of EGFR in vitro (Fig. 4), it does not appear to form a stable complex with EGFR, as expected, when co-expressed in cells (Fig. 5). In contrast, D1051A RPTP-{kappa} does form a stable complex with EGFR, which can be readily detected by Western analysis (Fig. 5). These data demonstrate that EGFR is a substrate for RPTP-{kappa} in intact cells.

Dephosphorylation of Endogenous EGFR by RPTP-{kappa} in Primary Human Keratinocytes—We next examined the effect of RPTP-{kappa} on tyrosine phosphorylation of endogenous EGFR in primary human keratinocytes. Adenovirus-mediated overexpression of RPTP-{kappa} in primary human keratinocytes significantly reduced EGF-induced EGFR tyrosine 1068 phosphorylation (Fig. 6). Expression of catalytically inactive 2C/S-RPTP-{kappa}, with cysteine to serine mutation in both PTP catalytic domains, increased both basal and EGF-induced EGFR tyrosine 1068 phosphorylation (Fig. 6). This increased EGFR tyrosine phosphorylation likely reflects dominant negative activity of catalytically inactive 2C/S-RPTP-{kappa} (12, 13). Reduction of EGFR tyrosine phosphorylation observed with expression of RPTP-{kappa} was specific, because adenovirus-mediated overexpression of RPTP-µ, which is most structurally related to RPTP-{kappa}, did not reduce EGF-induced EGFR tyrosine phosphorylation in human keratinocytes (Fig. 7).


Figure 6
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  		FIGURE 6.
Overexpression of wild-type RPTP-{kappa} reduces EGF-induced EGFR tyrosine phosphorylation in human keratinocytes. Primary human keratinocytes were infected with either empty (Ept), wild-type ({kappa}-wt), or catalytically inactive mutant His-tagged RPTP-{kappa} ({kappa}-mt) adenovirus. Two days after infection, cells were treated with vehicle (–, Veh) or EGF (+, 25 ng/ml) for 10 min at 37 °C. Whole cell lysates were analyzed for EGFR phosphotyrosine 1068 (pY-1068), total EGFR, and His-RPTP-{kappa} by Western blot. Data are means ± S.E. of fluorescent band intensities quantified by STORM as described under"Experimental Procedures." n = 4; *, p < 0.05 versus empty vector. Inset shows representative Western blot.

 


Figure 7
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  		FIGURE 7.
Co-expression of RPTP-µ with EGFR does not alter EGFR tyrosine phosphorylation in human keratinocytes. Primary human keratinocytes were infected with EGFR expression vector and either empty or RPTP-µ adenovirus. Two days after infection, the cells were treated with vehicle or EGF (10 ng/ml) for 5 min at 37 °C. Whole cell lysates were analyzed by Western blot using antibodies to EGFR phosphotyrosine 1068 (pY-1068), total EGFR, and RPTP-µ. Results are means ± S.E. of fluorescent band intensities quantified by STORM as described under"Experimental Procedures." n = 4. Inset shows representative Western blot. CTRL, control.

 
siRNA-mediated Knockdown of RPTP-{kappa} Increases EGFR Tyrosine Phosphorylation in Primary Human Keratinocytes—Transfection of RPTP-{kappa} siRNA was analyzed to determine the effect of reduced RPTP-{kappa} on EGFR tyrosine phosphorylation. RPTP-{kappa} siRNA specifically reduced RPTP-{kappa} mRNA by 60% (Fig. 8A), whereas it did not reduce mRNA levels of related RPTPs, RPTP-beta, RPTP-{delta}, RPTP-µ, and RPTP-{zeta} in confluent primary human keratinocytes (Fig. 8A). The RPTP-{kappa} protein level was reduced by 70% (Fig. 8B). Reduction of endogenous RPTP-{kappa} by siRNA increased EGFR basal tyrosine phosphorylation in a dose-dependent manner (Fig. 9A). In addition, reduction of endogenous RPTP-{kappa} by siRNA significantly potentiated EGF-stimulated EGFR tyrosine 1068 phosphorylation (Fig. 9B).


Figure 8
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  		FIGURE 8.
siRNA-mediated knockdown of RPTP-{kappa} in human keratinocytes. A, RPTP-{kappa} siRNA specifically reduces RPTP-{kappa} mRNA. Primary human keratinocytes were transfected with synthetic double-stranded control (CTRL) or RPTP-{kappa} siRNA (90 nM). Two days post-transfection, total RNA was isolated from confluent keratinocytes, and transcripts for the indicated RPTPs were quantified by real-time RT-PCR. Results are means ± S.E. of three experiments. *, p < 0.05 versus control siRNA. B, following transfection of RPTP-{kappa} siRNA, as described above, whole cell lysates were prepared and analyzed by Western blot using antibodies to RPTP-{kappa} and beta-actin. Data are means ± S.E. of fluorescent band intensities quantified by STORM as described under"Experimental Procedures." n = 3. *, p < 0.05 versus control siRNA. Inset shows representative Western blot.

 
RPTP-{kappa} Knockdown Potentiates EGF-induced Erk Activation in Primary Human Keratinocytes—One of the major downstream effectors of EGFR is Erk mitogen-activated protein kinase. Because reduction of endogenous RPTP-{kappa} potentiates basal and EGF-induced EGFR activation, we investigated the effect of RPTP-{kappa} knockdown on Erk activation. siRNA-mediated RPTP-{kappa} knockdown resulted in a 2-fold increase of basal phosphorylation of p44 and p42 Erk, in primary human keratinocytes (Fig. 9C). Similarly, RPTP-{kappa} knockdown resulted in a further 2-fold increase of EGF-induced phosphorylation of Erk (Fig. 9C).

RPTP-{kappa} Inhibits Keratinocyte Growth—In culture, growth of human keratinocytes slows and eventually stops as the cells reach confluency. Because keratinocyte growth is EGFR-dependent (19, 20), we examined RPTP-{kappa} expression as a function of culture confluency. We found that RPTP-{kappa} mRNA expression was relatively low in subconfluent keratinocytes and increased markedly when keratinocytes became confluent (Fig. 10A). These data indicate that increased expression of RPTP-{kappa} is associated with reduced keratinocyte growth.

To examine whether the level of RPTP-{kappa} can influence keratinocyte growth, RPTP-{kappa} was overexpressed in subconfluent keratinocyte cultures with low endogenous RPTP-{kappa} and the rate of proliferation determined. Both empty and RPTP-{kappa} adenovirus-treated keratinocytes displayed slow growth during the first two days after treatment, which is typical for human keratinocyte seeded at low density. Empty adenovirus-treated keratinocytes exhibited accelerated growth during days three and four post-treatment, reaching 80–90% confluency at day four (Fig. 10B). In contrast, RPTP-{kappa} adenovirus-treated keratinocytes showed no significant proliferation during days three and four post-treatment (Fig. 10B). During the course of the experiment, keratinocyte viability, determined by trypan blue exclusion, was >95% for both empty and RPTP-{kappa} adenovirus-treated keratinocytes.


Figure 9
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  		FIGURE 9.
siRNA-mediated knockdown of RPTP-{kappa} increases basal and potentiates EGF-induced EGFR tyrosine phosphorylation and Erk activation in human keratinocytes. A, human keratinocytes were transfected with the indicated concentrations of control (CTRL) or RPTP-{kappa} siRNA. Two days post-transfection, whole cell lysates were analyzed by Western blot for EGFR phosphotyrosine 1068 (pY-1068) and total EGFR. Results are means ± S.E. of fluorescent band intensities quantified by STORM as described under"Experimental Procedures." n = 2; *, p < 0.05 versus control siRNA. Inset shows representative Western blot. B, human keratinocytes were transfected with 90 nM control or RPTP-{kappa} siRNA. Two days post-transfection, the cells were treated with vehicle (Veh) or EGF (5 ng/ml) for 10 min at 37 °C. Whole cell lysates were analyzed by Western blot for EGFR phosphotyrosine 1068 (pY-1068) and total EGFR. Data are means ± S.E. of fluorescent band intensities quantified by STORM as described under"Experimental Procedures." n = 3; *, p < 0.05 versus control siRNA. Inset shows representative Western blot. C, human keratinocytes were transfected with 90 nM control or RPTP-{kappa} siRNA. Two days post-transfection, the cells were treated with vehicle (Veh) or EGF (5 ng/ml) at 37 °C for 30 min. Whole cell lysates were analyzed by Western blot for phospho- and total Erk. Results are means ± S.E. of fluorescent band intensities quantified by STORM as described under"Experimental Procedures." n = 3; *, p < 0.05 versus control siRNA. Inset shows representative Western blot.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We demonstrated that RPTP-{kappa} directly dephosphorylates EGFR in vitro and increased RPTP-{kappa} expression reduces basal and ligand-stimulated EGFR tyrosine phosphorylation, whereas reduced RPTP-{kappa} expression increases EGFR tyrosine phosphorylation. Regulation of EGFR tyrosine phosphorylation is complex and occurs at multiple levels, including ligand activation, internalization, recycling, biosynthesis, and dephosphorylation (21, 22). Several different PTPs have been demonstrated to reduce EGFR phosphorylation at various stages following ligand activation (14–18). To our knowledge, however, RPTP-{kappa} is unique among RPTPs in its ability to regulate both basal (i.e. in the absence of exogenous ligand) and ligand-activated EGFR tyrosine phosphorylation. In addition, RPTP-{kappa} is unique in its ability to regulate downstream EGFR function, including Erk activation and cellular proliferation.


Figure 10
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  		FIGURE 10.
RPTP-{kappa} expression is increased in confluent human keratinocytes and inhibits keratinocyte growth. A, human keratinocytes were seeded at 2500 cells/cm2, and RPTP-{kappa} mRNA levels were quantified by real-time RT-PCR 3 days (Subconfluent) and 7 days (Confluent) after seeding. Results are means ± S.E. n = 3; *, p < 0.05. B, human keratinocytes were seeded at 2500 cells/cm2 and 24 h later treated with either empty (open circles) or RPTP-{kappa} adenovirus (closed circles). The cells were harvested at the indicated times post-infection and counted by hemocytometer. Results are means ± S.E. n = 3; *, p < 0.05.

 
Human keratinocytes were found to express 13 of 21 known RPTPs. We tested the five most abundantly expressed RPTPs for their ability to reduce basal EGFR tyrosine phosphorylation. Among the five RPTPs tested, only RPTP-{kappa} was able to significantly reduce basal EGFR tyrosine phosphorylation. It is possible that other RPTPs, which we did not study, act in concert with RPTP-{kappa} to reduce EGFR phosphorylation. We found that RPTP-{kappa} preferentially dephosphorylated tyrosines 1068 and 1173, compared with tyrosine 992. This observation further supports the concept that EGFR is acted upon by multiple RPTPs. In this regard, both RPTP LAR, and RPTP-{sigma} have been shown to reduce EGFR phosphorylation (18, 23). However, whether either RPTP acts directly on EGFR, or which phosphotyrosine residue(s) either RPTP affects remains to be determined.

Human keratinocytes, similar to many other cell types in culture, cease proliferation in response to cell-cell contacts. Although the detailed mechanism of contact inhibition remains elusive, interactions among adhesion molecules on the surface of adjoining cells plays a pivotal role. The extracellular domains of many RPTPs contain adhesion molecule-like sequences, leading to the proposal that RPTP functions may be regulated, at least in part, by cell-cell contacts (24). In fact, RPTP-{kappa} and RPTP-µ have been shown to possess adhesion properties that can mediate cell-cell and cell-matrix communication (25, 26). Interestingly, membrane-associated PTP activity is increased up to 10-fold in contacted-inhibited cells and harvested at high density, compared with proliferating cells at low density, whereas tumor cells, which are not subjected to contact-inhibition, do not show density-dependent increase in membrane-associated PTP activity (27, 28). Furthermore, RPTP-µ, RPTP-beta, and DEP-1 have been shown to be up-regulated as a function of increased cell density in culture (24, 28, 29). In the current study, we demonstrated that RPTP-{kappa} levels also increase as a function of confluence in cultured human keratinocytes. This finding is consistent with the reported increased expression of RPTP-{kappa} with increased confluence in SK-BR-3 human mammary carcinoma cells (30).

In human keratinocytes, overexpression or reduced expression of RPTP-{kappa} decreased or elevated EGFR tyrosine phosphorylation, respectively. These changes in EGFR tyrosine phosphorylation were mirrored by alterations in the level of Erk activation. Thus, RPTP-{kappa} appears to regulate a key downstream EGFR effector through regulation of EGFR tyrosine phosphorylation. Erk is a critical mediator of keratinocyte proliferation and survival (31). Therefore, the ability of increased RPTP-{kappa} levels to reduce Erk activation is expected to reduce keratinocyte growth. Indeed, we found that raising the level of RPTP-{kappa} in subconfluent keratinocytes resulted in near complete inhibition of growth. Under these conditions, increased expression of RPTP-{kappa} did not alter the level of EGFR. These data suggest that the ratio of RPTP-{kappa} to EGFR, rather than the absolute level of either RPTP-{kappa} or EGFR, is an important determinant of EGFR functionality. A number of epithelial tumors are characterized by hyperactivation of EGFR. Hyperactivation can result from EGFR mutation or overexpression. However, our data suggest that reduced expression of RPTP-{kappa} also allows hyperactivation of EGFR in the absence of any alteration of EGFR itself. Whether reduced expression of RPTP-{kappa} occurs in any human epithelial cancers is currently under investigation.

RPTP LAR and RPTP DEP-1 have been shown to regulate insulin receptor and hepatocyte growth factor receptor signaling, respectively (32, 33). DEP-1 has also been reported to be involved in dephosphorylation of platelet-derived growth factor receptor in a site-specific manner (34). Given that RPTP-{kappa} dephosphorylates EGFR, raises the possibility that regulation of receptor protein-tyrosine kinases (RPTKs) by RPTPs may be a widespread mechanism of control. If so, further elucidation of which RPTPs act on which RPTKs may lead to important new insights into RPTP function and RPTK regulation. The possibility that a single RPTP may regulate more than one RPTK provides the possibility for a novel mechanism of cross-talk among seemingly distinct receptor-mediated signaling pathways. Whether or not RPTP-{kappa} dephosphorylates other RPTKs in addition to EGFR must await further investigation.

An additional mechanism by which RPTP-{kappa} participates in cross-talk between distinct receptor-mediated signaling pathways has recently been described (35). The transforming growth factor-beta (TGF-beta) receptor complex possesses intrinsic serine threonine kinase activity, which phosphorylates and thereby activates transcription factors Smad2/3. Smad2/3 mediate many cellular responses to TGF-beta (36). TGF-beta is a powerful inhibitor of epithelial cell growth and has previously been reported to induce RPTP-{kappa} in association with growth inhibition in the HaCaT keratinocyte cell line (37). Recently, TGF-beta induction of RPTP-{kappa} has been demonstrated to reduce ligand activation of EGFR and participate in TGF-beta-dependent growth reduction in breast cancer cell lines (35). Thus, RPTP-{kappa} emerges as a critical regulator of epithelial cell growth by setting the balance between the proliferative EGFR and anti-proliferative TGF-beta pathways. Of note is that TGF-beta responsiveness is lost in many epithelial cancers (38, 39). This loss may reduce tonic growth inhibition exerted by TGF-beta-induced RPTP-{kappa} on EGFR-mediated growth and thereby shift the balance toward more rapid growth in transformed cells. It will be of interest to determine RPTP-{kappa} levels in epithelial tumors, which have lost TGF-beta responsiveness.


   FOOTNOTES
 
* This work was supported in part by the Babcock Endowment. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Dept. of Dermatology, University of Michigan Medical School, Medical Science I, Rm. 6447, 1150 W. Medical Ctr. Dr., Ann Arbor, MI 48109-0609. Tel.: 734-763-1469; Fax: 734-647-0076; E-mail: dianemch{at}umich.edu.

2 The abbreviations used are: PTK, protein-tyrosine kinase; PTP, protein-tyrosine phosphatase; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; RPTP-{kappa}, receptor type protein-tyrosine-phosphatase-{kappa}; RPTK, receptor protein tyrosine-kinase; RPTP, receptor type PTPs; CHO, Chinese hamster ovary; TGF-beta, transforming growth factor-beta; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Axel Ullrich (Max-Planck Institute for Biochemistry, Germany) for pRK5 EGFR expression vector, Dr. H. Saito (Dana-Farber Cancer Institute) for human RPTP-{kappa}, RPTP-{zeta}, RPTP-beta, and RPTP-{delta} cDNA, Mr. Keith Wanzeck for technical support, and Laura VanGoor and Diane Fiolek for graphic and administrative support.



   REFERENCES
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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
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