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Originally published In Press as doi:10.1074/jbc.M310401200 on December 16, 2003

J. Biol. Chem., Vol. 279, Issue 11, 10765-10775, March 12, 2004
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Mutual Regulation of Protein-tyrosine Phosphatase 20 and Protein-tyrosine Kinase Tec Activities by Tyrosine Phosphorylation and Dephosphorylation*

Naohito Aoki{ddagger}§, Shuichi Ueno¶, Hiroyuki Mano¶, Sho Yamasaki||, Masayuki Shiota**, Hitoshi Miyazaki**, Yumiko Yamaguchi-Aoki{ddagger}{ddagger}, Tsukasa Matsuda{ddagger}, and Axel Ullrich{ddagger}{ddagger}

From the {ddagger}Department of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan, Divisions of Functional Genomics, Cardiology and Hematology, Jichi Medical School, Kawachi-gun, Tochigi 329-0498, Japan, ||Molecular Genetics, Chiba University Graduate School of Medicine, Chiba 260-8670, Japan, **Gene Research Center, University of Tsukuba, Ibaraki 305-8572, Japan, and {ddagger}{ddagger}Max Planck Institute for Biochemistry, Department of Molecular Biology, D-82152 Martinsried, Germany

Received for publication, September 22, 2003 , and in revised form, December 5, 2003.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
PTP20, also known as HSCF/protein-tyrosine phosphatase K1/fetal liver phosphatase 1/brain-derived phosphatase 1, is a cytosolic protein-tyrosine phosphatase with currently unknown biological relevance. We have identified that the nonreceptor protein-tyrosine kinase Tec-phosphorylated PTP20 on tyrosines and co-immunoprecipitated with the phosphatase in a phosphotyrosine-dependent manner. The interaction between the two proteins involved the Tec SH2 domain and the C-terminal tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20, which were also necessary for tyrosine phosphorylation/dephosphorylation. Association between endogenous PTP20 and Tec was also tyrosine phosphorylation-dependent in the immature B cell line Ramos. Finally, the Tyr-281 residue of PTP20 was shown to be critical for deactivating Tec in Ramos cells upon B cell receptor ligation as well as dephosphorylation and deactivation of Tec and PTP20 itself in transfected COS7 cells. Taken together, PTP20 appears to play a negative role in Tec-mediated signaling, and Tec-PTP20 interaction might represent a negative feedback mechanism.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Protein-tyrosine phosphatases (PTPs)1 are a large and structurally diverse family of enzymes that catalyze the dephosphorylation of tyrosine-phosphorylated proteins (1, 2). Biochemical and kinetic studies have documented that Cys and an Asp residues in the catalytic domain are essential for the PTP activity. PTPs have been shown to participate as either positive or negative regulators of signaling pathways in a wide range of physiological processes, including cellular growth, differentiation, migration, and survival (1, 2). Despite their important roles in such fundamental cellular processes, the mechanisms by which PTPs exert their effects are largely not understood.

PTP20 (3), which is also known as hematopoietic stem cell fraction (HSCF) (4), PTP-K1 (5), fetal liver phosphatase 1 (6), and brain-derived phosphatase 1 (7), comprises the PEST family of PTPs together with PTP-PEST and PEP PTP and was originally isolated by screening a PC12 cDNA library. Overexpression of PTP20 in PC12 cells results in a more rapid and robust neurite outgrowth in response to nerve growth factor treatment, suggesting that PTP20 is involved in cytoskeletal reorganization (3). Mostly consistent with this observation, overexpression of a dominant negative mutant of fetal liver phosphatase 1 in K562 hematopoietic progenitor cells results in an inhibition of cell spreading and substrate adhesion in response to phorbol ester (6). Recently, through yeast two-hybrid screening the proline, serine, threonine phosphatase-interacting protein (PSTPIP) and PSTPIP2 have been identified to be specific in vivo substrates for HSCF, because the phosphotyrosine (Tyr(P)) level of PSTPIP1 is significantly enhanced by coexpression of the catalytically inactive mutant (Cys to Ser) of PTP20 (8, 9). PSTPIP is tyrosine-phosphorylated both in BaF3 cells and in v-Src-transfected COS cells and is shown to be co-localized with the cortical actin cytoskeleton, lamellipodia, and actin-rich cytokinetic cleavage furrow (8), strongly supporting the idea that PTP20/HSCF is a potential regulator of cytokinesis. PSTPIP also interacts with the C-terminal part of the cytosolic protein-tyrosine kinase (PTK) c-Abl, serves as a substrate for c-Abl, and can bridge interactions between c-Abl and PTP20 with the dephosphorylation of c-Abl by PTP20 (10). It has also been reported that PTP20 associates with the negative Src-family kinase regulator Csk via its Src homology 2 (SH2) domain and two putative sites of tyrosine phosphorylation of the phosphatase (11). This association is thought to allow Csk and PTP20 to synergistically inhibit Src-family kinase activity by phosphorylating and dephosphorylating negative and positive regulatory tyrosine residues, respectively.

Regarding post-translational regulation of the PEST family PTPs, it has been documented that phosphorylation of an N-terminal serine residue, which is well conserved in all members of the PEST PTP family, by protein kinase A results in the inhibition of its catalytic activity (12). In addition to proline, serine, and threonine residues in the C-terminal PEST domain of PTP20, a large number of tyrosine residues exist in that region, suggesting the possibility that PTP20 is tyrosine-phosphorylated. Indeed, previous studies reveal that PTP20/HSCF becomes tyrosine-phosphorylated by constitutively active forms of Lck and v-Src kinases in transfected cells (8, 11) even though the physiological relevance of tyrosine phosphorylation on PTP20 remains unclear.

In this study we addressed the question of PTP20 regulation with special emphasis on the relevance of tyrosine phosphorylation and its biological impact. Through co-expression with nonreceptor PTKs we found that Tec kinase strongly tyrosine-phosphorylated the catalytically inactive form of PTP20 and that Tec physically interacted with PTP20 in a tyrosine phosphorylation-dependent manner in transfected COS7 cells. Further analyses with a variety of mutants of PTP20 and Tec revealed that C-terminal tyrosine residues of PTP20 and the Tec SH2 domain were necessary in the regulation of respective state of phosphorylation. Ectopic expression of PTP20 in human immature Ramos B cells resulted in suppression of B-cell receptor-induced c-fos promoter activity. Moreover, we determined that tyrosine 281 of PTP20 played a role in the dephosphorylation activity of PTP20 against both Tec and PTP20 itself. Our findings suggest a negative feedback mechanism that mutually controls the tyrosine phosphorylation of Tec and PTP20 and regulates Tec activity and B cell receptor (BCR) signaling.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—Antibodies to hemagglutinin (HA) epitope (Y-11), phosphotyrosine (PY99), glutathione S-transferase (GST) (Z-5), Src (SRC2), Lck (2102), JAK2 (M-126), JAK3 (C-21), Csk (C-20), and ZAP70 (LR) were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA). Antibodies to Tec, Itk, Btk, and Bmx were as described previously (13). Antibody to PTP20 was prepared by immunizing rabbits with the N-terminal peptide of PTP20 (MSRQSDLVRSFLEQQEARDH), to which a cysteine residue was added to the C terminus, coupled to keyhole limpet hemocyanin (14). Anti-human IgM antibody (Fab')2 fragment was obtained from Southern Biotechnology Associates (Birmingham, AL). All other reagents were from Sigma unless otherwise noted.

Plasmid Construction—The pSR-based expression vectors for Tec wild-type (WT), Tec kinase mutant, TecY187F, TecY518F, and Tec proteins lacking each subdomain were described previously (15, 16). pEBG plasmids (17) encoding each subdomain of Tec to express the GST-tagged proteins were previously described (18). HA epitope tagging to PTP20 at its N terminus and subsequently all the mutations (cysteine to serine, aspartic acid to alanine, and tyrosine to phenylalanine) in PTP20 were carried out by PCR-based strategy. To express GST-tagged PTP20 in mammalian cells, full-length PTP20 (amino acids 2–453), PTP catalytic domain (amino acids 2–308), and the C-terminal noncatalytic PEST domain (amino acids 271–453) were amplified by PCR and ligated into pEBG vector via the BamHI site. All the plasmids newly constructed were confirmed by sequencing. Expression plasmids for rat Csk and mouse JAK2 were generous gifts from Drs. M. Okada (Osaka University, Japan) and J. N. Ihle (St. Jude Children's Research Hospital, Memphis, TN), respectively. Expression plasmids for mouse Src, Lck, Itk, Btk, Bmx, ZAP-70, and JAK3 were described elsewhere.

Cell Culture and Transfection—COS7 cells were cultured in Dulbecco's modified Eagle's medium (high glucose, Sigma) supplemented with 10% fetal calf serum. Ramos cells (American Type Culture Collection, Manassas, VA) were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal calf serum. Upon transfection experiments COS7 cells were inoculated at a density of 4 x 105 cells/6-cm dish and grown overnight in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Expression plasmids were transfected into the cells by the modified calcium phosphate precipitation method (19). After incubation under 3% CO2, 97% air for 18 h, the transfected cells were washed with phosphate-buffered saline twice and cultured in fresh Dulbecco's modified Eagle's medium containing 10% fetal calf serum for another 24 h under humidified 5% CO2 and 95% air.

Cell Lysis, Immunoprecipitation, GST Pull-down, and Western Blotting—The transfected cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 5 mM EDTA, 150 mM NaCl, 10 mM sodium phosphate, 10 mM sodium fluoride, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, and 10 µg/ml leupeptin. Lysates were directly subjected to immunoblotting, immunoprecipitation with the indicated antibodies plus protein G- or Protein A-Sepharose beads (Amersham Bioscience), or precipitation with GSH-Sepharose beads (Amersham Bioscience). Proteins in the immunoprecipitates and precipitates were further analyzed by immunoblotting with the indicated antibodies. The protein bands were visualized with an enhanced chemiluminescence (ECL) detection kit (Amersham Bioscience) and light capture system (AE-6962, ATTO, Tokyo, Japan).

c-fos Promoter Assay—Ramos cells (1 x 107/experiment) were subjected to electroporation with 2 µg of the pfos/luc reporter plasmid (20) plus 10 µg of expression plasmids for PTP20 or its mutants. Five hours after transfection cells were incubated for 5 h in the absence or presence of antibodies to human IgM (10 µg/ml). Luciferase activity was measured with the use of the dual luciferase assay system (Promega, Madison, WI).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Tec Is a Potent Regulator of PTP20—Although PTP20 has been shown to be a substrate of v-Src (8) and constitutively active Lck (11), the physiological relevance of PTP20 tyrosine phosphorylation remains unknown. Northern blot analysis revealed that PTP20 was abundantly expressed in spleen, suggesting a role in the immune system (data not shown). Therefore, it was reasoned that other PTKs of immune cells might be involved in PTP20 regulation by tyrosine phosphorylation. To examine this possibility HA-tagged PTP20 was co-expressed with various cytosolic PTKs including Src and Lck in COS7 cells. We used a catalytically inactive form of PTP20 for this experiment because autodephosphorylation activity of PTP20 has been previously reported (8). Cells were lysed, PTP20 was immunoprecipitated with anti-HA antibody, and the immune complexes were subjected to SDS-PAGE and immunoblotting with anti-phosphotyrosine antibody. As shown in Fig. 1A, PTP20 was tyrosine-phosphorylated by Src and Lck and co-immunoprecipitated with proteins with 56 and 60 kDa, likely corresponding to Lck and Src, respectively. In the case of ectopic Lck expression, endogenous Src seemed to be included in the immune complex, as suggested by the presence of a 66-kDa phosphotyrosine-containing band. PTP20 was slightly tyrosine-phosphorylated by Csk and co-immunoprecipitated with a faintly tyrosine-phosphorylated 70-kDa band, which seemed unlikely to be Csk. JAK2 but not JAK3 also tyrosine-phosphorylated PTP20 and appeared to be co-immunoprecipitated with PTP20. Most notably, PTP20 was strongly tyrosine-phosphorylated by Tec and co-immunoprecipitated with a heavily tyrosine-phosphorylated protein of 74 kDa and other minor proteins of 120 and 35 kDa. Based on the molecular mass, the 74-kDa protein was likely to represent Tec. Itk, another member of Tec/Btk family, also tyrosine-phosphorylated PTP20 to a lesser extent and was co-immunoprecipitated, whereas related PTKs Btk and Bmx did not tyrosine phosphorylate PTP20 and were not co-immunoprecipitated. Because all the transfected PTKs were obviously expressed as compared with mock transfectant (Fig. 1, panel B), it was suggested that Tec tyrosine-phosphorylated PTP20 with the greatest efficiency.



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  		FIG. 1.
Tyrosine phosphorylation of PTP20 by various PTKs. A, COS7 cells were transiently transfected with HA-PTP20 C/S together with either Lck, Src, JAK2, JAK3, Tec, Itk, Btk, Bmx, Csk, or ZAP70. Cells were lysed, and PTP20 was immunoprecipitated (IP) with anti-HA antibody followed by immunoblotting (WB) with anti-phosphotyrosine antibody (PY99 ({alpha}pY)). The same membrane was reprobed with anti-HA antibody after stripping. B, an aliquot of the cell lysates was immunoblotted with the indicated antibodies to confirm substantial expression of each PTK.

 
Tec Is a Potential Substrate of PTP20—To examine the relationship between PTP20 and Tec in more detail, Tec was co-transfected with WT or a catalytically inactive C/S form of PTP20 into COS7 cells, and either PTP20 or Tec was immunoprecipitated followed by immunoblotting with anti-phosphotyrosine antibody. When HA-PTP20 WT was expressed, no phosphorylated bands were visible in both anti-HA and anti-Tec immunoprecipitates, possibly due to dephosphorylation activity of PTP20 against both Tec and itself (Fig. 2). Two major bands with 74 and 50 kDa in the anti-HA and anti-Tec immune complexes were detected with anti-phosphotyrosine antibody only when the PTP20 C/S mutant was co-transfected with Tec. Reprobing with anti-Tec and anti-HA antibodies clearly revealed that the bands represent Tec and HA-PTP20. No phosphorylation of Tec was observed when Tec alone was introduced into COS7 cells, suggesting that the interaction between Tec and PTP20 was required for Tec phosphorylation and possibly activation. These results suggest that PTP20 is a substrate of Tec and that Tec is also a substrate of PTP20.



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  		FIG. 2.
Tyrosine phosphorylation-dependent interaction of PTP20 with Tec. Tec cDNA was transiently introduced into COS7 cells together with either empty vector (mock), HA-PTP20 WT, or C/S and lysed. PTP20 or Tec was immunoprecipitated either with anti-HA (left panels) or anti-Tec (right panels) antibody, respectively. The immunoprecipitates (IP) were separated by SDS-PAGE followed by immunoblotting (WB) sequentially with the indicated antibodies. The bands corresponding to Tec and PTP20 are indicated by arrows. In either case expression of Tec or HA-PTP20 was confirmed using aliquots of total cell lysates (TCL) by immunoblotting (lowest panels). {alpha}pY, anti-phosphotyrosine antibody.

 
Phosphotyrosine-dependent Interaction between PTP20 and Tec—Tec is composed of several distinct domains including pleckstrin homology (PH), Tec homology (TH), SH3, SH2, and kinase (KD) domains (Fig. 3, panel A). All of these domains are necessary for full function of Tec under physiological conditions (15, 16). To examine which domains are involved in interaction with PTP20, Tec mutants each lacking one of the domains were co-transfected with the catalytically inactive form of PTP20 into COS7 cells. A kinase mutant as well as two mutants (Y187F and Y518F) where tyrosine residues were replaced by phenylalanines were also included. Cells were lysed, and PTP20 was immunoprecipitated followed by immunoblotting with anti-phosphotyrosine antibody. PTP20 was tyrosine-phosphorylated by the Y187F mutant as well as mutants lacking PH, TH, and SH3 domains to a similar extent as compared with Tec WT (Fig. 3, panel B). As expected, the Y518F mutant, which is missing the autophosphorylation site for Tec activation, and the inactive mutant of a kinase mutant could not tyrosine phosphorylate PTP20. Interestingly, the {Delta}PH mutant could tyrosine phosphorylate PTP20 but was not co-immunoprecipitated with PTP20. Most strikingly, the {Delta}SH2 mutant could not tyrosine phosphorylate PTP20 and was not co-immunoprecipitated with PTP20. When a membrane on which aliquots of total cell lysates were blotted was probed with anti-phosphotyrosine antibody, it was revealed that co-expression of the Tec {Delta}SH2 mutant and PTP20 resulted in no tyrosine phosphorylation on both molecules and that the Tec {Delta}PH mutant tyrosine-phosphorylated (Fig. 3, panel C). Tec SH2 domain-dependent interaction with PTP20 was further investigated by co-transfecting the PTP20 C/S mutant with plasmids encoding GST fusion proteins of Tec domains in the presence or absence of Tec into COS7 cells. Cell lysates were subjected to pull-down experiments with GSH-Sepharose beads. Precipitates were separated by SDS-PAGE followed by immunoblotting with the indicated antibodies. In the absence of full-length Tec co-expression, no substantial binding of PTP20 to any of the Tec domains was apparent (Fig. 3, panel D). In contrast, in the presence of full-length Tec, phosphorylated PTP20 bound to only the SH2 domain of Tec. Given that co-expression of Tec should result in marked tyrosine phosphorylation of PTP20 in COS7 cells, these data indicate that the PTP20-Tec interaction is mediated predominantly by the SH2 domain of Tec and phosphotyrosine residues of PTP20.



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  		FIG. 3.
Tec SH2 domain is essential for both tyrosine phosphorylation of PTP20 and association of Tec with PTP20. A, schematic organization of mouse Tec into PH, TH, SH3, SH2, and kinase (KD) domains. B, COS7 cells were transiently transfected with either empty vector (mock) or HA-PTP20 C/S together with the indicated Tec mutants. Cells were lysed, and HA-PTP20 was immunoprecipitated (IP) followed by immunoblotting (IB) with anti-phosphotyrosine antibody (PY99 ({alpha}pY)). The same membrane was sequentially reprobed with anti-Tec and anti-HA antibodies after stripping. C, aliquots of the total cell lysates (TCL) were separated by SDS-PAGE followed by immunoblotting with indicated antibodies. D, COS7 cells were transiently transfected with pEBG empty vector (GST) or bearing each of Tec domains (PH, TH, SH3, SH2, and KD) in the absence or presence of Tec plasmid. Cells were lysed, and GST fusion proteins were precipitated by GSH-Sepharose beads followed by immunoblot analysis with anti-phosphotyrosine (pY) antibody. The same membrane was sequentially reprobed with indicated antibodies. Expression of PTP20 and Tec was confirmed using aliquots of total cell lysates (TCL) by immunoblotting as indicated.

 
Next, we tried to identify the binding site(s) for Tec in PTP20. Because the interaction of Tec with PTP20 was mediated by the Tec SH2 domain, potential tyrosine residues of phosphorylation were first taken into consideration. There are 13 tyrosine residues (Tyr-62, Tyr-68, Tyr-86, Tyr-101, Tyr-144, Tyr-192, Tyr-244, Tyr-281, Tyr-285, Tyr-303, Tyr-354, Tyr-381, Tyr-419) in the PTP20 sequence, and all the residues are perfectly conserved among human and mouse orthologs (Fig. 4). We focused our attention on the tyrosine residues Tyr-281, Tyr-285, Tyr-303, Tyr-354, Tyr-381, and Tyr-419 located in the C-terminal PEST domain of PTP20, and 6 residues were individually mutated.



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  		FIG. 4.
Sequence alignment of PTP20 with its human (brain-derived phosphatase 1 (BDP1)) and mouse (HSCF) orthologs. The 13 conserved tyrosine residues are boxed and numbered based on the amino acid sequence of PTP20. PTP catalytic domains are indicated by gray shading.

 
First, the mutants were tested for the extent of tyrosine phosphorylation by Tec in transfected COS7 cells. Total cell lysates were subjected to anti-phosphotyrosine blotting. Fig. 5, panel A, demonstrates that the PTP20 mutants (Y281F, Y303F, Y354F, Y381F) in which Tyr-281, Tyr-303, Tyr-354, and Tyr-381 were individually mutated exhibited dramatic reduction in tyrosine phosphorylation levels, whereas no apparent reduction for Y285F and Y419F was observed. Combinational mutation of Tyr-281, Tyr-303, Tyr-354, and Tyr-381 totally abolished tyrosine phosphorylation of PTP20. In keeping with these data, anti-phosphotyrosine blotting also demonstrated that tyrosine phosphorylation of Tec was concomitantly reduced. This observation was further extended by GST pull-down experiments using the Tec SH2 domain. COS7 cells were then transfected with PTP20 YF variants together with Tec and Tec-Tec SH2, as outlined in Fig. 3, panel C. Mutation of either Tyr-281, Tyr-303, Tyr-354, or Tyr-381 of PTP20 resulted in reduced binding capacity of PTP20 to the Tec SH2 domain, and again, such binding was completely abrogated by substituting all the tyrosine residues (Fig. 5, panel B). Together these data clearly indicate that four tyrosine residues in the C-terminal non-catalytic region of PTP20 are involved in not only binding to the Tec SH2 domain but also in the phosphorylation and subsequent activation of Tec.



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  		FIG. 5.
Specific tyrosine residues of PTP20 are necessary for tyrosine phosphorylation of Tec and association with Tec SH2 domain. A, HA-PTP20 C/S or its YF (tyrosine to phenylalanine substitution) mutants as indicated were co-transfected into COS7 cells with Tec. Aliquots of total cell lysates (TCL) were immunoblotted (WB) with anti-phosphotyrosine ({alpha}pY) antibody. The same membrane was sequentially reprobed with anti-Tec and -HA antibodies. B, COS7 cells were co-transfected with expression plasmids for HA-PTP20 C/S or its YF mutants, Tec, and GST-Tec-SH2 domain. Cells were lysed, and GST-Tec-SH2 domain was precipitated with GSH-Sepharose beads followed by immunoblot analysis by sequential probing with anti-phosphotyrosine, anti-HA, and anti-GST antibodies. Expression of nearly the same amounts of PTP20 was confirmed by immunoblotting of aliquots of total cell lysates with anti-HA antibody.

 
We asked whether the C-terminal non-catalytic region of PTP20 was enough for phosphorylation and activation of Tec. To this end, PTP20 deletion mutants lacking either an N-terminal catalytic or a C-terminal non-catalytic segment were made, but the resultant constructs could not be expressed in COS7 cells, although comparable amounts of transcripts were detected (data not shown). To solve this problem, the N-terminal PTP domain and the C-terminal PEST domain were inserted into pEBG vector and were expressed as GST fusion proteins in COS7 cells. These pEBG plasmids encoding the PTP domain and full length of PTP20 C/S mutant and the PEST domain of PTP20 were co-transfected into COS7 together with Tec. Anti-phosphotyrosine blotting documented that Tec was highly tyrosine-phosphorylated with the full-length but not the PTP domain of the PTP20 C/S mutant (Fig. 6, panel A), supporting previous data shown in Fig. 5, where the C-terminal part of PTP20 was essential for tyrosine phosphorylation of Tec. Interestingly, the presence of the PEST domain of PTP20 caused tyrosine phosphorylation of PTP20, but the extent was lower than in the presence of the full-length PTP20 C/S mutant. Equivalent expression of each construct was confirmed by Western blotting with anti-Tec and anti-GST antibodies. To further examine the involvement of the PEST domain, lysates were precipitated with GSH-Sepharose beads followed by immunoblotting with anti-phosphotyrosine antibody. A phosphorylated 74-kDa band, which was shown to be Tec by immunoblotting, was co-precipitated with full-length PTP20 C/S mutant, whereas the PTP domain alone could not capture Tec (Fig. 6, panel B). A faint tyrosine-phosphorylated band with the same mobility of 74 kDa that co-precipitated with the PEST domain appeared to be Tec but could not be detected by our anti-Tec antibody presumably due to sensitivity. These results suggest that the PEST domain of PTP20 is necessary but not sufficient for not only hyperphosphorylation and activation of, but also association with Tec.



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  		FIG. 6.
Both PTP catalytic and PEST domains of PTP20 are involved in maximal phosphorylation of Tec and association with Tec. Tec was co-transfected with either empty pEBG vector (-) or that bearing the PTP20 catalytic domain (PTP), full-length PTP20 (Full), or the non-catalytic PEST domain of PTP20 (PEST). A, aliquots of total cell lysates (TCL) were subjected to immunoblotting with anti-phosphotyrosine antibody ({alpha}pY, upper panel). The same membrane was reprobed with a mixture of anti-Tec and anti-GST antibodies. B, remaining cell lysates were precipitated with GSH-Sepharose beads and processed as mentioned above. The bands corresponding to individual products are indicated by arrows.

 
Negative Regulatory Roles of PTP20 in BCR Signaling—All the experiments documented above were conducted in transfected COS7 cells. To demonstrate a physiological relevance of the PTP20-Tec interaction, evidence of such an association in non-transfected cells was required. To this end we selected human Ramos immature B cells, because it has been reported that they express relatively high amounts of endogenous Tec (21). As shown above, interaction of PTP20 with Tec is mediated by tyrosine phosphorylation of PTP20, and PTP20 has autodephosphorylation activity, implying that it would be difficult to detect a phosphotyrosine-dependent interaction of PTP20 with other molecules including Tec endogenously. To overcome this experimental difficulty, protein-tyrosine phosphorylation was induced in Ramos cells by treatment with pervanadate (POV). Cells were starved for 16 h in serum-free medium and then either left unstimulated or treated with 0.1 mM POV for 30 min and lysed. Cell lysates were immunoprecipitated with either anti-phosphotyrosine antibody or anti-Tec antibody. Our PTP20-specific antibody could not be used due to its inability in immunoprecipitation experiments. In anti-phosphotyrosine immunoprecipitates, specific bands with 74 and 50 kDa corresponding to human Tec and PTP20 were detected only upon POV treatment (Fig. 7). A tyrosine-phosphorylated band with 50 kDa in the anti-Tec immunoprecipitates was readily detected by the anti-PTP20 antibody but only when cells received POV pretreatment (Fig. 7). These results indicate that endogenous Tec and PTP20 interact with each other in a phosphotyrosine-dependent manner in Ramos B cells.



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  		FIG. 7.
Tyrosine phosphorylation-dependent interaction of endogenous PTP20 with endogenous Tec in Ramos B cells. Ramos cells were treated with 0.1 mM POV for 15 min at 37 °C, lysed, and subjected to immunoprecipitation with either anti-phosphotyrosine ({alpha}pY) or anti-Tec antibody. The immunoprecipitates (IP) were immunoblotted (WB) by anti-phosphotyrosine antibody. The same membranes were sequentially reprobed with anti-PTP20 and -Tec antibodies. The bands corresponding to Tec and PTP20 are indicated by arrowheads.

 
Although upstream regulators such as cytokine receptors, lymphocyte surface antigens, G protein-coupled receptors, receptor type PTKs, or integrins for Tec in blood cells including Ramos B cells have been relatively well investigated (13, 20, 22–26), only limited information regarding downstream regulators of Tec has been available so far. If the data obtained in transfected COS7 cells are true, PTP20 would be thought to play a negative regulatory role in Tec-mediated signaling. To examine this, either the PTP20 WT, the inactive C/S mutant, or another form of catalytically inactive mutant D/A was transiently co-transfected with the pfos/luc reporter plasmid into Ramos cells, because the promoter of the c-fos proto-oncogene is activated in response to BCR cross-linking in the cells. Cells were either left unstimulated or treated with anti-human IgM F(ab')2 fragments for 5 h. Cell lysates were assayed for luciferase activity. BCR cross-linking induced a marked activation of the c-fos promoter (Fig. 8). Expression of PTP20 WT totally inhibited BCR-induced activation of the c-fos promoter as well as its basal activity, whereas only about 20% inhibition of the promoter activation was observed in the co-expression of catalytically inactive forms of PTP20, strongly indicating that PTP20 is a negative regulator of BCR-Tec-c-fos signaling.



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  		FIG. 8.
Negative role of PTP20 in BCR signaling. Ramos cells (1 x 107) were subjected to electroporation with 2 µg of the pfos/luc reporter plasmid together with 10 µg of pcDNA3 vector (mock) or bearing PTP20 WT, C/S, or D/A mutant. Five hours after transfection cells were incubated for an additional 5 h in the absence (open bars) or presence (closed bars) of anti-IgM (ab') (10 µg/ml). Cells lysates were then assayed for luciferase activity. Data are expressed as mean ± S.D. of triplicate determinations.

 
Tyrosine Phosphorylation of PTP20 by Tec Modulates Its Catalytic Activity against Tec as Well as Itself—We demonstrated that specific tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 of PTP20 could be phosphorylated by Tec and served as Tec binding sites (Fig. 5). To further investigate physiological relevance of PTP20 tyrosine phosphorylation, substitution of the tyrosine residues with phenylalanine in PTP20 WT was performed. The YF mutants of HA-PTP20 WT were co-transfected with Tec and the PTP20 C/S mutant without epitope tagging, and effects on the extent of tyrosine phosphorylation on Tec were analyzed by anti-phosphotyrosine blotting. As shown in Fig. 9A, substitution of Tyr-281 with phenylalanine (Y281F) resulted in dramatic loss of PTP20 dephosphorylation activity against Tec. On the other hand, Tec could be dephosphorylated by Y303F, Y354F, and Y381F to nearly the same extent by PTP20 WT. The PTP20 Y281F/Y303F/Y354/F381F mutant in which 4 tyrosine residues were substituted by phenylalanine also exhibited apparently no dephosphorylation activity against Tec. Equivalent expression of HA-PTP20 was confirmed by immunoblotting (lowest panel). Next, the autodephosphorylation activity of the YF mutants of HA-PTP20 WT was assessed by co-transfecting Tec and-PEST encoding the GST-PTP20 PEST domain into COS7 cells, as GST-PTP20 PEST alone became tyrosine-phosphorylated in the presence of Tec (Fig. 6). Cells were lysed and GST-PTP20 PEST was precipitated with GSH-Sepharose beads followed by anti-phosphotyrosine blotting. Again, PTP20 Y281F as well as PTP20 Y281F/Y303F/Y354/F381F showed no dephosphorylation activity against GST-PTP20 PEST, whereas PTP20 Y303F, Y354F, and Y381F as well as PTP20 WT could dephosphorylate GST-PTP20 PEST (Fig. 9B). These YF mutants also were transfected into Ramos B cells, and c-fos promoter activity was assayed after BCR ligation. Ectopic expression of PTP20 Y281F and Y281F/Y303F/Y354/F381F mutants still inhibited c-fos promoter activity (about 50%, relative to mock transfectants), but the extent was significantly lower than that of WT as well as other YF mutants. These results strongly suggest that phosphorylation of Tyr-281 on PTP20 is essential for expression of catalytic activity against not only Tec but also PTP20 itself in transfected COS7 cells as well as in Ramos B cells, although other tyrosine residues, Tyr-303, Tyr-354, and Tyr-381, are also phosphorylated by Tec.



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  		FIG. 9.
Tyrosine 281 is critical for in vivo phosphatase activity of PTP20. A, COS7 cells were co-transfected with Tec, PTP20 C/S, and HA-PTP20 WT or its YF mutants. HA-PTP20 C/S was also included as a negative control. Cells were lysed, and Tec was immunoprecipitated with anti-Tec antibody. The immunoprecipitates (IP) were separated by SDS-PAGE followed by immunoblotting (WB) with indicated antibodies. Expression of HA-PTP20 was confirmed using aliquots of total cell lysates (TCL) with anti-HA antibody. {alpha}pY, anti-phosphotyrosine antibody. B, COS7 cells were transfected as above, but PEST-encoding GST-PTP20 PEST domain (GST-PEST) in place of PTP20 C/S was included. Cell lysates were subjected to precipitation with GSH-Sepharose beads and immunoblotted with the indicated antibodies. Expression of HA-PTP20 was confirmed using aliquots of total cell lysates (TCL) with anti-HA antibody. C, Ramos cells were transfected by electroporation with 2 µg of the pfos/luc reporter plasmid together with 10 µg of pcDNA3 vector (mock) or bearing PTP20 WT or its YF mutant and processed as described in legend to Fig. 8.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Many signaling pathways triggered by PTKs can be potentially modulated by PTPs in a negative or positive manner under cellular context. In some cases phosphorylation on the tyrosine residues of PTPs themselves can modulate their catalytic activities. For example, SH2 domain-containing PTP SHP-2 is tyrosine-phosphorylated upon stimulation by a variety of growth factors (27–29) and cytokines (30–35). Once SHP-2 becomes tyrosine-phosphorylated, their catalytic activity might be increased and modulated its own tyrosine phosphorylation level by autodephosphorylation activity (36, 37). It also has been reported that tyrosine phosphorylation of PTP1B upon insulin and epidermal growth factor treatment causes reduction in its catalytic activity, thereby enhancing apparent insulin receptor- and epidermal growth factor receptor-mediated signaling pathways (38, 39). Thus, tyrosine phosphorylation of PTPs appeared to be critical for the regulation of their biological functions.

Among the PEST family PTPs, PTP20 is an only member that gets phosphorylated on tyrosine residues, whereas no tyrosine phosphorylation of other members, PTP-PEST and PTP-PEP, has been reported. In the present study, we clearly demonstrated that PTP20 was tyrosine-phosphorylated by a cytosolic Tec kinase. As previously reported for phosphorylation of PTP20 by constitutively active Src family kinases (8, 11), the catalytically inactive form of PTP20 was found to be tyrosine-phosphorylated to a greater extent by Tec, whereas apparently no phosphorylation on PTP20 WT was obvious, possibly due to its autodephosphorylation activity. Src and Lck indeed tyrosine-phosphorylated PTP20, but the extent of tyrosine phosphorylation of PTP20 by Tec was shown to be the greatest (Fig. 1). Moreover, related Itk did tyrosine-phosphorylate PTP20 to a lesser extent, but Btk and Bmx did not (Fig. 1). These results suggest that Tec kinase tyrosine phosphorylates PTP20 more specifically and preferentially than Src family kinases and its related kinases do.

Without ectopic PTP20 expression, tyrosine phosphorylation of Tec kinase was not detected in transfected COS7 cells (Fig. 2). Although co-expression of PTP20 WT did not induce tyrosine phosphorylation of Tec, the catalytically inactive C/S variant of PTP20 caused tyrosine phosphorylation of Tec and co-immunoprecipitated with Tec. These results strongly suggest that a dominant-negative effect of PTP20 C/S expression on Tec tyrosine phosphorylation seems to be unlikely and, rather, that Tec was possibly autophosphorylated and further activated by interacting with PTP20 and then was immediately dephosphorylated and deactivated by PTP20, which might also be activated through interaction with Tecin a tyrosine phosphorylation-dependent manner. A deletion of the Tec SH2 domain abrogated tyrosine phosphorylation of Tec as well as PTP20 and association between Tec and PTP20 (Fig. 3). Likewise, substitution of individual tyrosine residues Tyr-281, Tyr-303, Tyr-354, and Tyr-381 with phenylalanines of PTP20 reduced not only tyrosine phosphorylation of Tec and PTP20 itself but also association of PTP20 with the Tec SH2 domain (Fig. 5). Substitution of all the four tyrosine residues (Fig. 5) as well as a deletion of the C-terminal non-catalytic segment (Fig. 6) completely abolished those events, and the C-terminal segment alone partially induced Tec tyrosine phosphorylation (Fig. 6), supporting the idea that phosphotyrosine-dependent interaction between PTP20 and Tec is essential for determining a mutual state of phosphorylation and activation. Taken together, we propose a working hypothesis of tyrosine phosphorylation-dependent interaction between PTP20 and Tec kinase (Fig. 10).



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  		FIG. 10.
Working hypothesis for interaction of PTP20 with Tec. I, upon stimuli or ectopic expression of Tec, Tec becomes tyrosine-phosphorylated and activated through autophosphorylation and other PTK catalytic activity. In turn, Tec phosphorylates tyrosine residues (Tyr-281, Tyr-303, Tyr-354, Tyr-381) on PTP20. II, phosphorylated PTP20 associates with Tec SH2 domain of remaining inactive Tec, thereby activating the Tec kinases. Interaction of Tec with PTP20 increases a pool of activated Tec and PTP20. III, activated PTP20 by phosphorylation then dephosphorylates Tec as well as PTP20 itself. Note that free of phosphorylated tyrosine 281 from association with Tec SH2 domain might be necessary for expression of PTP20 dephosphorylation activity. IV, finally, both Tec and PTP20 return to basal and inactive states.

 
PTPs exhibit elaborate substrate specificity in vivo. This specificity can be achieved at two levels. First, the phosphatase catalytic domain itself displays an intrinsic specificity for its substrate. However, the affinity between the catalytic domain and its substrate is often low. Actually, the PTP domain of the catalytically inactive PTP20 alone could not capture a potential substrate Tec kinase (Fig. 6). A further enhancement of the specificity is achieved by protein-protein targeting; the Tec SH2 domain and phosphorylated tyrosine residues on PTP20 could enhance the interaction between the two molecules. In Ramos B cells, we could detect tyrosine phosphorylation-dependent interaction between PTP20 and Tec only when cells were treated with pervanadate (Fig. 7). In this case, however, apparent binding might have resulted from a sole interaction of phosphorylated tyrosines of PTP20 C-terminal with the Tec SH2 domain and, therefore, underestimated because vanadate can get into the catalytic pocket of PTP20 reversibly and inhibit interaction between the PTP domain segment of PTP20 and tyrosine-phosphorylated Tec kinase. Upon physiological stimulation both PTP20 catalytic domain-Tec phosphotyrosine(s) and PTP20 phosphotyrosine-Tec SH2 domain bindings could play an essential role.

Most interestingly, tyrosine phosphorylation of PTP20 appears to regulate its catalytic activity against Tec and PTP20 itself. Among the tyrosine residues phosphorylated by Tec kinase, tyrosine 281 might be critical for dephosphorylation activity of PTP20 in transfected COS7 cells as well as in Ramos B cells (Fig. 9). In the case of ectopic expression in COS7 cells, substitution of the Tyr-281 nearly abolished dephosphorylation activity against both PTP20 and Tec (Fig. 9, A and B). On the other hand Y281F as well as Y281F/Y303F/Y354/F381F mutants exhibited reduced, but still ~50% dephosphorylation activity as compared with mock transfectants when expressed in Ramos B cells (Fig. 9C), suggesting that other direct or indirect mechanisms to regulate PTP20 activity are involved in dephosphorylation and deactivation of Tec in the cells. However, we cannot exclude the possibility that phosphorylation on serine and threonine residues rich in the C-terminal region of PTP20 might affect catalytic activity of PTP20, as PTP20 can be regulated under the control of follicle-stimulating hormone in rat ovarian granulosa cells, where no tyrosine phosphorylation on PTP20 was observed (14).

It has been reported that constitutively active Lck phosphorylates tyrosine residues 354 and 381 on PTP20, which are in turn recognized by the Csk SH2 domain (11). In that report it was also documented that mutation of both the tyrosine residues on PTP20 caused no changes in catalytic activity by in vitro phosphatase assay. We have also showed that PTP20 was tyrosine-phosphorylated by Lck and Src and was associated with the PTKs (Fig. 2). However, neither the SH2 nor the SH3 domain of Lck was shown to be involved in the association with PTP20 (data not shown). Recently, another cytosolic protein-tyrosine kinase c-Abl also was shown to phosphorylate PTP20 and in turn to be dephosphorylated by PTP20 (10). Although PTP20-Tec and PTP20-cAbl interactions seem to be analogous, association between PTP20 and c-Abl is indirect, and PSTPIP, which is also a substrate of PTP20, instead serves as an adapter by bridging PTP20 to c-Abl. In contrast, association between PTP20 and Tec kinase seems to be direct, and involvement of adaptor molecules such as PSTPIPs is unlikely because the Tec SH2 domain alone could capture tyrosine-phosphorylated PTP20 (Fig. 3D) and, consistently, substitution of tyrosine residues on PTP20 dramatically reduced the mutual binding (Fig. 5B). These imply that PTP20 might be differentially tyrosine-phosphorylated by Lck, Tec, and c-Abl kinases depending on cellular context.

The Tec kinase was initially isolated from mouse liver (40) and was subsequently shown to be expressed in many tissues, including spleen, lung, brain, and kidney (41). Four Tec-related PTKs, including Btk (42, 43), Itk (also known as Emt or Tsk) (44–46), Bmx (47), and Txk (or Rlk) (48, 49), have also been molecularly cloned. Tec and the related kinases can be activated by cytokine receptors, lymphocyte surface antigens, G protein-coupled receptors, receptor type PTKs, or integrins (13, 20, 22–26). However, little is known about how the inactivation of Tec kinase is achieved. In this study, we have showed that PTP20 is a potential negative regulator in Tec-mediated signaling pathway and that the Tec SH2 domain is essential for the negative regulation by PTP20. Itk, another member of Tec family, might also be regulated by PTP20 in T cells in a similar fashion,2 whereas Btk and Bmx seem not to interact with PTP20 (Fig. 1). Recently, the Tec SH2 domain has been shown to bind to Dok-1, which is tyrosine-phosphorylated by Tec, causing inhibition of BCR-mediated c-fos promoter activation (18). Another publication has demonstrated that a docking protein, BRDG1, binds to the Tec SH2 domain and acts downstream of Tec in a positive fashion in BCR signaling (50). Thus, the Tec SH2 domain might differentially participate in BCR signaling in a positive or negative way.

PTP D1, which comprises another subfamily of cytosolic PTPs, is shown to be a potential regulator and effector for not only Bmx/Etk kinase but also Tec kinase (51). The PH but not SH2 domain of Bmx/Etk is involved in the interaction with the central portion (residues 726–848) of PTP D1, and such binding is phosphotyrosine-independent, unlike PTP20-Tec interaction. Interaction between Bmx/Etk and PTP D1 stimulates the kinase activity of Bmx/Etk, resulting in an increased phosphotyrosine content in both proteins. Although it is obvious that PTP D1 is a substrate of Bmx/Etk and Tec, PTP D1 appears not to dephosphorylate the kinases. Rather, PTP D1 is a positive regulator in Bmx/Etk- and Tec-mediated signaling pathway leading to STAT3 activation. By co-transfection experiments, we observed that PTP36, which belongs to the same PTP subfamily as PTPD1, was tyrosine-phosphorylated by Tec kinase (data not shown). Thus, Tec-mediated signaling could be negatively or positively regulated by interacting with PTPs.

In conclusion, PTP20 appears to play a negative role in the Tec-mediated, in particular in BCR, signaling pathways and the tyrosine phosphorylation-dependent interaction between Tec and PTP20 might form a negative feedback loop. To our knowledge this is the first report demonstrating that tyrosine phosphorylation-dependent interaction between PTK and PTP is relevant for their mutual state in some cellular context.


   FOOTNOTES
 
* This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan (to N. A. and T. M.). 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

§ To whom all correspondence should be addressed: Dept. of Applied Molecular Biosciences, Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Fax: 81-52-789-4128; E-mail: naoki{at}agr.nagoya-u.ac.jp.

1 The abbreviations used are: PTP, protein-tyrosine phosphatase; HSCF, hematopoietic stem cell fraction; PSTPIP, proline, serine, threonine phosphatase-interacting protein; PTK, protein-tyrosine kinase; BCR, B cell receptor; SH2, Src homology 2; SH3, Src homology 3; HA, hemagglutinin; GST, glutathione S-transferase; WT, wild type; ECL, enhanced chemiluminescence; PH, pleckstrin homology; TH, Tec homology; POV, pervanadate. Back

2 S. Yamasaki and N. Aoki, unpublished data. Back



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