INTRODUCTION

Top Abstract Introduction Materials & Methods Results Discussion References

Mitogenic stimulation of cells by extracellular factors is often mediated by transmembrane tyrosine kinase receptors or receptors that associate with cytoplasmic tyrosine kinases. The signaling pathways generated by many of these receptors are well characterized (23). In contrast to the tyrosine kinase receptors, the receptor signaling pathways for transforming growth factor  (TGF-) and the many TGF--related factors have only recently been characterized (12, 24). TGF- and TGF--related factors are secreted proteins which mediate their activities through transmembrane serine/threonine kinase receptors. Ligand-induced activation of these receptors and signaling leads to potent growth inhibition and gene expression responses. Two type I and two type II receptors form the signaling TGF- receptor complex at the cell surface, in which the type II receptors (TRII) are constitutively active and autophosphorylated, and the type I receptors (TRI) require phosphorylation by TRII for activation (12, 24).

Several proteins have been shown to associate with TGF- receptors. Smad2 and Smad3, which act as effectors of TGF- signaling, can associate with the receptor complex and are phosphorylated by TRI. Once dissociated, they are translocated as a complex with Smad4/DPC4 into the nucleus, where they function as transcriptional activators (11, 24, 33). Another receptor-associated protein is TRIP-1, which interacts with and is phosphorylated by TRII (8) and contains five WD-40 repeats (40). WD-40 repeats are minimally conserved sequences of approximately 40 amino acids that typically end in tryptophan-aspartate (WD) and are thought to mediate protein-protein interactions (40). Since TRIP-1 is largely composed of WD-40 repeats, it is possible that other WD-40 repeat proteins may bind to serine/threonine kinase receptors. The association of WD-40 repeat proteins may then allow them to play a role in signaling by the serine/threonine kinase receptors. WD-40 repeats have been identified in a variety of proteins (40), including the B subunit of the serine/threonine protein phosphatase 2A (PP2A).

PP2A is one of the major, albeit poorly understood, serine/threonine phosphatases which regulates several processes, including signaling (43) and cell cycle progression (9, 44). PP2A exists as a dimeric core of a catalytic (C) and a structural (A) subunit or as a trimeric complex with a regulatory subunit (B), of which there are several forms. B and B contain five WD-40 repeats (40), whereas B'/B56 (38) and B" (46) are structurally unrelated and lack WD-40 repeats (45, 46). B regulates the catalytic activity of PP2A, and this activity has been implicated in cell cycle control (21, 35). The differential interactions of these regulatory B subunits with the AC core enzyme suggest a complex pattern of regulation, which may explain the various functions of PP2A in growth control.

Because B has been implicated in cell cycle control (9, 21, 35) and has WD-40 repeats like TRIP-1 does, we analyzed the physical and functional interactions between B and TGF- receptors. In this report, we demonstrate that the B regulatory subunit of PP2A interacts with the cytoplasmic domains of type I TGF- receptors and is a direct target for their kinase activity. The growth inhibitory activity of B is regulated by TGF- receptors and cooperates with the direct antiproliferative effect of the TGF- receptors. Thus, the association of the WD-40 repeat B subunit of PP2A with serine/threonine kinase receptors results in a functional interaction of TGF- receptor signaling with B and may be important for our understanding of how PP2A activity is regulated.

	MATERIALS AND METHODS

Top Abstract Introduction Materials & Methods Results Discussion References

In vitro translation and association with GST fusion proteins. To generate ³⁵S-labeled B or B' in vitro, 2 µg of pRK7-B, i.e., the B cDNA subcloned in pRK7 (18), or pRK5-B', i.e., the B' cDNA in pRK5 (18) or pRK5-TRIP-1 (7), was used to transcribe the cDNAs from the SP6 promoter (Promega kit). The transcripts were translated and ³⁵S labeled by using the TNT rabbit reticulocyte lysate kit (Promega) while being incubated at 30°C for 2 h. The translation mixture was then preadsorbed to glutathione S-transferase (GST) protein bound to glutathione-Sepharose beads in binding buffer (50 mM Tris-HCl [pH 7.5], 120 mM NaCl, 2 mM EDTA, 0.1% Nonidet P-40 [NP-40]) for 1 h at 4°C. The supernatant was then added to glutathione-Sepharose beads with 1 µg of purified GST protein or with 1 µg of the cytoplasmic domains of Tsk7L/R1 (amino acids 146 to 509), TRI/R4 (amino acids 152 to 503), or TRII (amino acids 194 to 567) fused to GST (GST-R1, GST-R4, or GST-TRII) and incubated for 1 h at 4°C. Following adsorption, the glutathione beads with their adsorbed proteins were washed four times with binding buffer and specifically bound proteins were eluted twice with 50 µl of 50 mM Tris (pH 8.0)-5 mM reduced glutathione (42). Eluted proteins were diluted into loading buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The ³⁵S-labeled bands were then detected by autoradiography.

Chemical cross-linking. PP2A subunits and complexes, i.e. B, ABC, and ABC, were expressed in baculovirus-infected SF9 insect cells and purified as described previously (25). The His₆-tagged cytoplasmic domains of the TGF- receptors, i.e., TRII (amino acids 194 to 567), TRI/R4 (amino acids 152 to 503), and Tsk7L/R1 (amino acids 153 to 509), were also expressed in SF9 cells and affinity purified with Co²⁺-Sepharose beads and eluted from beads in 10 mM sodium phosphate (pH 8.0)-150 mM NaCl-100 mM imidazole. One microgram of B, ABC, or ABC was incubated with 1 µg of His₆-tagged cytoplasmic domain of a receptor, and the protein interactions were stabilized by chemical cross-linking with 2 mM bismaleimidohexane (Pierce)-50 mM MOPS (morpholinepropanesulfonic acid; pH 7.0)-150 mM NaCl-1 mM EDTA for 90 min at 4°C. Protein samples were diluted into loading buffer and separated by SDS-PAGE. Following transfer of samples to nitrocellulose, Western blotting was performed by using anti-B antibody, and immunodetection was carried out by enhanced chemiluminescence (ECL) and autoradiography.

In vitro phosphorylation. One hundred nanograms of His₆-tagged cytoplasmic domain of Tsk7L/R1 was incubated alone, with 200 ng of B, or with 200 ng of ABC in a kinase reaction mixture (27 mM HEPES [pH 7.4], 4 mM MnCl₂, 40 mM nitrophenylphosphate, 10 µCi of [-³²P]ATP per nmol). Reaction mixtures were incubated for 30 min at 30°C, proteins were separated by SDS-PAGE, and ³²P-labeled bands were visualized by autoradiography.

In vivo association of B with the receptor cytoplasmic domains. COS-1 cells were transfected by using Lipofectamine (Gibco-BRL) with expression plasmids while the total amount of plasmid DNA was kept at 15 µg. To express B, we used 10 µg of pCMV5-B, i.e., pCMV5 (2) containing the coding sequence for B with a C-terminal FLAG tag, whereas the receptor cytoplasmic domains were expressed from 5 µg of pRK5 encoding His₆-tagged kinase-active or -inactive cytoplasmic domains of Tsk7L/R1 (amino acids 153 to 509), TRI/R4 (amino acids 152 to 503), or TRII (amino acids 194 to 567). To determine the effect of full-length TRII on the association of B with the cytoplasmic domain of R4, 0, 1, or 10 µg of pRK5-TRII (RII) was cotransfected with TRI/R4 and pCMV5-B as described above, except that total DNA was kept at 25 µg. At 24 h after transfection, the cells were washed and labeled with 100 µCi of [³⁵S]Met-Cys in Dulbecco modified Eagle medium (DMEM)-10% fetal bovine serum (FBS) for 16 h. The cells were washed with phosphate-buffered saline (PBS) and lysed with 10 mM Na phosphate (pH 8.0)-150 mM NaCl-0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride (PMSF)-1 mM benzamidine. Cleared lysates were then incubated with Co²⁺-Sepharose beads for 1 h at 4°C, and the beads were washed with 10 mM Na phosphate (pH 8.0)-150 mM NaCl-25 mM imidazole (Sigma). Specifically bound proteins, i.e. His₆-tagged cytoplasmic domains with associated proteins, were eluted with 10 mM Na phosphate (pH 8.0)-150 mM NaCl-100 mM imidazole. The proteins were separated by SDS-PAGE and transferred to nitrocellulose, and receptor-associated B was identified by Western blotting with anti-B antibody, ECL, and autoradiography.

Cell surface biotinylation and coimmunoprecipitation of receptors with B. COS-1 cells in 10-cm-diameter plates were transfected by using Lipofectamine with 16 µg of plasmid DNA. To express B, we used 8 µg of pCMV5-B, i.e., pCMV-5 containing the coding sequence of B with a C-terminal FLAG tag, whereas full-length type I receptors were expressed from 8 µg of pRK5 encoding C-terminally Myc-tagged Tsk7L/R1 or TRI/R4. At 36 h after transfection, the cells were washed with PBS and incubated with 1 mg of normal human serum-LC-biotin at 4°C for 45 min. The cells were then washed and incubated for 10 min with 50 mM glycine in PBS-0.5 mM MgCl₂ (pH 7.8). The cells were lysed with 50 mM HEPES (pH 7.5)-1% NP-40, and cleared lysates were incubated with anti-FLAG antibody and protein A-Sepharose beads. The beads were then washed with a series of buffers, starting with 12.5 mM K phosphate (pH 7.4)-0.6 M NaCl, and then 10 mM Tris (pH 8.3)-0.1% SDS-0.05% NP-40-0.3 M NaCl, followed by 12.5 mM K phosphate (pH 7.5)-0.6 M NaCl and 12.5 mM K phosphate (pH 7.5)-0.3 M NaCl, and finally 20 mM Tris (pH 8.3). B and coprecipitating proteins were eluted with gel sample buffer, analyzed by SDS-PAGE, and transferred to nitrocellulose, and the biotinylated bands were identified by using horseradish peroxidase-conjugated streptavidin and ECL.

In vivo phosphorylation of N-terminally truncated B. COS-1 cells were cotransfected by using Lipofectamine with pRK5-Myc-B#50-447, encoding B lacking N-terminal amino acids 1 to 49, with or without pRK5-TRII (TRII) and pRK5-R4 (R4). At 36 h after transfection, the cells were labeled with 1 mCi of [³²P]orthophosphate per ml for 12 h, incubated with 1 µM okadaic acid for 1 h, and then stimulated for 30 min with 400 pM TGF-. The cells were washed and lysed at 4°C in 27 mM HEPES (pH 7.4)-150 mM NaCl-0.5 mM EGTA-0.1% Triton X-100-1 µM okadaic acid-40 mM nitrophenylphosphate-100 µM orthovanadate-1 mM PMSF-1 mM benzamidine. ³²P-labeled B#50-447 was immunoprecipitated with anti-Myc 9E10 antibody and detected after SDS-PAGE and autoradiography of ³²P-labeled bands.

In vivo TGF- receptor phosphorylation. L17 cells, a highly transfectable Mv1Lu mutant cell line lacking functional TRI/R4 (48), were cotransfected by using Lipofectamine (Gibco-BRL) with 2 µg of the B expression plasmid pRK7-B, or the parental pRK7 plasmid, and 1 µg of pRK5-TRII-Flag (TRII) and pRK5-R4-Flag (R4). At 36 h after transfection, the cells were washed and labeled with 1 mCi of [³²P]orthophosphate in DMEM without phosphate-10% FBS for 4 h. The cells were stimulated for 30 min with 400 pM TGF- and washed and lysed at 4°C in 27 mM HEPES (pH 7.4)-150 mM NaCl-0.5 mM EGTA-0.1% Triton X-100-40 mM nitrophenylphosphate-100 µM orthovanadate-1 mM PMSF-1 mM benzamidine. Receptors were immunoprecipitated with anti-FLAG M2 antibody, purified proteins were separated by SDS-PAGE, and receptors were detected by autoradiography of ³²P-labeled bands.

PP2A assay. The expression plasmid pRK7-B or pRK7 were cotransfected with pHook2/neo (Invitrogen) into HaCaT cells, and pools of stably transfected cells were selected by using 750 µg of G418 (Sigma) per ml. The cells were then plated in six-well dishes and grown to 70% confluence in DMEM containing 10% FBS. After incubation with or without 400 pM TGF- for 10 min, the cells were placed on ice, washed, and lysed. The lysates were diluted to the same protein concentration, and equal aliquots were assayed for phosphatase activity for 10 min at 30°C in 20 mM MOPS (pH 7.0)-0.5-mg/ml BSA-0.5 mM dithiothreitol (DTT)-1-mg/ml [³²P]phosphorylase A (prepared by using the Gibco BRL phosphatase kit) in the presence or absence of 1 nM okadaic acid (Gibco-BRL). Reactions were terminated by adding 15% trichloroacetic acid and 2.5 mg of BSA per ml. Proteins were precipitated for 10 min at 4°C and pelleted by centrifugation for 5 min. ³²P released into the supernatant was measured by liquid scintillation counting. PP2A activity was defined as the phosphorylase A phosphatase activity inhibitable by 1 nM okadaic acid (10).

PAI-1- and cyclin A-luciferase assays in HaCaT cells. HaCaT cells (16) were grown to 40% confluence in six-well dishes. An 0.8-µg amount of pRK7-B or pRK7 was cotransfected with Lipofectamine with 0.4 µg of pRKgal and either 0.4 µg of p3TP-lux, for PAI-1-luciferase assays (47), or 0.4 µg of pCal2, for cyclin A-luciferase activity (14), and incubated at 37°C for 18 h. In parallel experiments, 0.8 µg of B or 0.4 µg of pRK5-Smad3 and 0.4 µg of pRK5-Smad4 were transfected with 0.5 µg of pCal2 and 0.5 µg of pSVgal and incubated at 37°C for 18 h. The medium was then replaced with DMEM-0.2% FBS with or without 100 pM TGF- and incubated for 24 h. Cleared lysates were prepared and assayed for luciferase (by using Promega's assay kit and a Monolight 2010 luminometer from Analytical Luminescence Laboratory) and -galactosidase (Tropix kit) activities. The luciferase activity was normalized against the -galactosidase activity as a measure of transfection efficiency.

PAI-1 protein production and DNA synthesis in transfected HaCaT cells, overexpressing B. To measure PAI-1 protein production, pools of HaCaT cells, transfected with pRK7 or pRK7-B, were grown to 50% confluence in six-well dishes in DMEM-10% FBS. The cells were washed with PBS and incubated in a mixture of DMEM without Met-Cys and 25 µCi of [³⁵S]Met per ml, with or without 100 pM TGF-, for 2 h at 37°C. The cells were washed, and extracellular matrix proteins were purified as described previously (22). ³⁵S-labeled proteins, including PAI-1, were separated by SDS-PAGE and detected by autoradiography. To measure DNA synthesis, HaCaT cells transfected with pRK7 or pRK7-B were plated in 24-well dishes and grown to 50% confluence. The cells were washed with PBS and incubated with 0.2% FBS with or without 100 pM TGF- for 20 h at 37°C. A 4-µCi/ml concentration of [³H]thymidine was added to the medium for 4 h, and the cells were washed and trypsinized. Trypsinized cells were collected on Whatman GF/C filters in a filtration apparatus, washed with PBS-10% trichloroacetic acid, and dried, and the radioactivity on the filters was measured by scintillation counting.

Cyclin A-luciferase assays in SW480.7 cells. SW480.7 cells (17, 49) were grown to 60% confluence in six-well dishes. An 0.8-µg amount of pCMV5-B or pRK7 was cotransfected by using Lipofectamine with 0.4 µg of pRK5-Smad3 and 0.4 µg of pRK5-Smad4 or pRK5 (as control), and 0.4 µg of pCal2 for cyclin A-luciferase activity (14). Transfected cells were incubated at 37°C for 18 h. Media were then replaced with DMEM-0.2% FBS with or without 100 pM TGF- and incubated for 24 h. Cleared lysates were prepared and assayed for luciferase and -galactosidase activities. The luciferase activity was normalized against the -galactosidase activity as a measure of transfection efficiency.

Cyclin A-luciferase assay in Mv1Lu cells. Confluent Mv1Lu cells trypsinized from a 100-mm-diameter plate were electroporated with 45 µg of plasmid DNA in DMEM by using 0.4-cm-gap cuvettes (Bio-Rad) at 960 µF and 350 V. The electroporated DNA contained 25 µg of pRK7-B or pRK7, 5 µg of pRK5-TRIDN (DN R4), which drives the expression of cytoplasmically truncated TRI/R4, 5 µg of pCal2, 5 µg of pRKgal, and 5 µg of pBluescript. Electroporated cells were allowed to recover for 4 h at 37°C in DMEM-10% FBS and then washed with PBS and incubated in the presence or absence of 100 pM TGF- in DMEM-0.2% FBS. After 48 h, cleared lysates were prepared and assayed for luciferase and -galactosidase activities.

	RESULTS

Top Abstract Introduction Materials & Methods Results Discussion References

In vitro association of the B subunit of PP2A with type I TGF- receptors. To evaluate the association of the WD-40 repeat protein B with serine/threonine kinase receptors, we first assessed its ability to directly interact with their cytoplasmic domains in vitro. Three receptors were tested for their ability to associate with B: the type II TGF- receptor (TRII) (31) and two type I receptors, TRI/R4 (15), which mediates growth inhibition and gene induction by TGF- in various cell types (3, 15), and Tsk7L/R1 (13), which is involved in TGF--mediated transdifferentiation of NMuMG cells (39) and responds to bone morphogenetic proteins when coexpressed with the bone morphogenetic protein type II receptor (32). ³⁵S-labeled, in vitro-translated B interacted with the cytoplasmic domains of the two type I receptors fused to (GST), but not with GST alone, and only minimally with the TRII cytoplasmic domain (Fig. 1A). In contrast, TRIP-1, which like B has five WD-40 repeats and associates with the TRII receptor (8), did not interact with the type I receptor cytoplasmic domain (Fig. 1B), consistent with our previous results (8). We therefore conclude that B specifically and directly associates with the type I receptor cytoplasmic domain in vitro.

View larger version (33K): [in this window] [in a new window]   FIG. 1.   In vitro association of B with type I receptors. (A) Association of in vitro-translated B with GST-receptor fusion proteins. In vitro-translated 35S-labeled B was incubated with glutathione-Sepharose-coupled GST (lane 2) or GST fused to the cytoplasmic domains of Tsk7L/R1 (lanes 3 and 5), TRI/R4 (lane 4), or TRII (lane 6). Bound proteins were eluted and separated by SDS-PAGE. Lane 1 shows 1/50 of the input 35S-labeled B used in each of the GST adsorption experiments. (B) Lack of interaction of TRIP-1 with the cytoplasmic domain of a type I receptor fused to GST (GST-R1) or with GST itself. (C) Gel electrophoretic analysis of the purified GST fusion experiments used in panel A and other experiments. Whereas the GST protein itself corresponded to a single band on the gel, the fusion proteins ran as several bands, the largest of which corresponded to the full-size proteins, and the smaller bands are presumably degradation products. (D) Covalent cross-linking of B in the ABC complex with the cytoplasmic domain of Tsk7L/R1. Purified ABC complex was incubated without cross-linking (lane 1) or was chemically cross-linked in the absence (lane 2) or presence (lane 3) of purified cytoplasmic domain of Tsk7L/R1. Cross-linked proteins were separated by SDS-PAGE and immunoblotted with anti-B antibody. The arrow points to the cross-linked complex of B with Tsk7L/R1 which is detected as an anti-B immunoreactive band. The arrowhead points to noncomplexed B. (E) Covalent cross-linking of B in the ABC complex with the cytoplasmic domain of Tsk7L/R1. Purified ABC was chemically cross-linked in the absence (lane 1) or presence (lane 2) of the His6-tagged cytoplasmic domain Tsk7L/R1. The arrow points to the cross-linked complex of B with Tsk7L/R1 which is detected as the anti-B immunoreactive band, and the arrowhead points to noncomplexed B. (F) In vitro-translated B' does not bind GST-receptor fusion proteins. In vitro-translated 35S-labeled B (lanes 1 to 3) and B' (lanes 4 to 6) were tested for their association with GST (lanes 1 and 4) or GST fusion proteins with the cytoplasmic domains of Tsk7L/R1 (lanes 2 and 5) or TRI/R4 (lanes 3 and 6) as described for panel A.

Type I receptor kinase-dependent association and phosphorylation of B. Type I receptors, like type II receptors, are serine/threonine kinases. Since the WD-40 repeat protein TRIP-1 interacts with higher affinity with the kinase-active than with the kinase-inactive type II receptor (8), we examined whether association of B with the type I receptor also depended on its kinase activity. As shown in Fig. 2A, B associated with the kinase-active cytoplasmic domain (lane 4) but not with a kinase-inactive point mutant (lane 3) and was detected as a 100-kDa, cross-linked anti-B immunoreactive band.

View larger version (35K): [in this window] [in a new window]   FIG. 2.   Interaction of B with the type I receptor cytoplasmic domain depends on its kinase activity and allows phosphorylation of B by the receptor kinase. (A) B interacts with kinase-active [ki(+)], not with kinase-inactive [ki()], Tsk7L/R1. B in the absence of the cytoplasmic domain of the receptor without (lane 1) or after (lane 2) chemical cross-linking is shown. B incubated with the kinase-inactive (lane 3) or kinase-active (lane 4) cytoplasmic domain of the receptor and stabilized by chemical cross-linking is also shown. The arrow points to the cross-linked complex of B with Tsk7L/R1 which is detected as the anti-B immunoreactive band, and the arrowhead points to noncomplexed B. Lanes 5 and 6 show that equal amounts of the kinase-negative or -positive cytoplasmic domains of Tsk7L/R1, purified as His6-tagged proteins, were used in lanes 3 and 4. (B) Tsk7L/R1 phosphorylates B. The purified cytoplasmic domain of Tsk7L/R1 was incubated alone (lane 1), with B (lane 2), or with ABC (lane 3) in a kinase reaction in the presence of [-32P]ATP. Proteins were separated by SDS-PAGE, and 32P-phosphorylated substrates were visualized by autoradiography. Besides B, a degradation product of B [B (degraded)] was also phosphorylated. The phosphorylated band above the B band is a protein that copurified with the receptor cytoplasmic domain. The gray arrowheads denote the positions of the A (upper) and C (lower) subunits, which were not phosphorylated. Lanes 4 to 6 show Western blots of purified and electrophoretically separated ABC (26), which was used in lane 3, with antibodies specific for A, B, or C, respectively.

Interaction of B with type I receptors in vivo. To determine whether B associates with the type I receptor in vivo, we coexpressed B with the type I receptors and assessed their interaction by using coimmunoprecipitation and Western blot analyses. To distinguish between B, the type I receptors, and the antibody heavy chain, which all migrate with the same mobility in SDS-PAGE, we coexpressed B with the cytoplasmic domains of the receptors preceded with an N-terminal methionine and a His₆ sequence. Purification of the receptor cytoplasmic domains by adsorption to Co²⁺-Sepharose thus allowed copurification of receptor-associated B. Subsequent Western blot analyses using anti-B antiserum showed that B associated with the cytoplasmic domains of the TRI and Tsk7L type I receptors (Fig. 3A, lanes 3 and 5) but did not interact with the kinase-inactive mutants of these receptors (Fig. 3A, lanes 2 and 4) nor with the type II receptor cytoplasmic domain (Fig. 3A, lanes 6 and 7). The A and C proteins were not detectable by using Western blot analyses or assays for phosphatase activity (data not shown), suggesting that receptor-bound B may not interact with the AC core enzyme or that low levels of associated AC may not be detectable. The low endogenous receptor levels and limitations in quality of the antibodies made detection of the association of endogenous B and type I receptors technically not feasible.

View larger version (26K): [in this window] [in a new window]   FIG. 3.   In vivo association of B with type I receptors. (A) In vivo association of B with type I receptors. B was expressed alone (lane 1) or coexpressed with His6-tagged kinase-inactive (lanes 2, 4, and 6) or kinase-active (lanes 3, 5, and 7) cytoplasmic domains of the type I receptor Tsk7L/R1 (lanes 2 and 3) or TRI/R4 (lanes 4 and 5) or the type II receptor TRII (lanes 6 and 7) in transfected COS-1 cells. His6-tagged cytoplasmic domains and associated proteins were purified on Co2+-Sepharose beads and, following elution, separated by SDS-PAGE, and B was detected by immunoblotting with anti-B antibody. (B) TRII coexpression enhances the association of B with TRI. B was expressed alone (lane 1) or was coexpressed with the His6-tagged cytoplasmic domain of TRI/R4 (lanes 2 to 4) in transfected COS-1 cells, in the absence (lane 2) or presence (lanes 3 and 4) of TRII (1 [lane 3] or 10 [lane 4] µg of transfected plasmid DNA). As in panel A, the proteins associated with TRI purified on Co2+-Sepharose beads were separated by SDS-PAGE and associated B was detected by immunoblotting. (C) Interaction of B with full-size type I receptor at the cell surface. The full-size type I receptor Tsk7L/R1 (lanes 2 and 3) or TRI/R4 (lanes 5 and 6) was expressed in COS-1 cells in the presence (lanes 3 and 6) or absence (lanes 2 and 5) of B in transfected cells. The corresponding cytoplasmically truncated Tsk7L/R1 (lane 1) or TRI/R4 (lane 4) was also coexpressed with B. Cell surface proteins were labeled by surface biotinylation of intact cells, and immunoprecipitations were carried out by using anti-B antibody, followed by visualization of coprecipitated, biotinylated proteins. The full-size glycosylated and unglycosylated type I receptor and an often-observed degradation product (degraded?) are indicated.

In vivo phosphorylation of N-terminally truncated B. Whereas the phosphorylation of B was readily demonstrated in vitro, it was much harder to define conditions that convincingly showed receptor-dependent phosphorylation of B in vivo. In the absence of okadaic acid, phosphorylation of B was not detected with or without overexpression of TGF- receptors. In contrast, in the presence of 1 µM okadaic acid, B was already highly phosphorylated in the absence of exogenous TGF- and this high level was only minimally enhanced by TGF- (data not shown). The lack of convincing evidence for B phosphorylation by TGF- receptors under our conditions may be due to the endogenous autocrine activation of the TGF- receptors, and the very transient nature of the phosphorylation may be due to rapid dephosphorylation of B by the core PP2A enzyme. We therefore analyzed the ability of the receptors to phosphorylate a mutant of B which lacks its N-terminal 49 amino acids. This small truncation abolished the interaction of B with the A and C subunits of PP2A (46a). As shown in Fig. 4, this B mutant is phosphorylated only when coexpressed with TGF- receptors. This observation demonstrates the ability of TGF- receptors to phosphorylate B in vivo. Our inability to convincingly demonstrate full-size B phosphorylation by the receptor may be due to its rapid association with and dephosphorylation by the PP2A core enzyme.

View larger version (32K): [in this window] [in a new window]   FIG. 4.   N-terminally truncated B is phosphorylated upon coexpression of TRI and TRII. Myc-epitope-tagged B, lacking amino acids 1 to 49, was coexpressed in the presence (+) or absence () of TRI/R4 and TRII (TRI/II) in transfected COS cells. The in vivo phosphorylation of this deletion mutant of B was assessed by in vivo 32P labeling followed by immunoprecipitation with anti-Myc antibody 9E10 and detection by SDS-PAGE and autoradiography (upper lanes). The lower lanes illustrate equal expression levels of the Myc-tagged B mutant as assessed by anti-Myc Western blotting of anti-Myc immunoprecipitated protein. Left lanes, without TRI/TRII expression; right lanes, with TRI/TRII expression.

B expression does not affect receptor phosphorylation. Since the B subunit can associate with the type I receptors and also can regulate PP2A activity through its association with the AC core enzyme (25), it is possible that B recruits the phosphatase to the activated receptor complex, which could then alter the phosphorylation state of the receptors. To test this, we coexpressed TRI/R4 and TRII with or without B and analyzed the in vivo ³²P phosphorylation levels of the receptors after TGF- stimulation. The level of phosphorylation of TRI/R4 or TRII, however, was not detectably altered with increased B expression (Fig. 5). In addition, no time-dependent alteration in the level of receptor phosphorylation was observed following TGF- stimulation (data not shown). Therefore, association of B with the receptors does not result in detectable overall changes in receptor phosphorylation, although differential phosphorylation of individual amino acids cannot be excluded.

View larger version (55K): [in this window] [in a new window]   FIG. 5.   Increased B expression does not alter the overall phosphorylation state of TGF- receptors. FLAG-tagged type II receptors (TRII) and type I receptor TRI/R4 (R4) were coexpressed with B in transfected L17 cells. The in vivo phosphorylation of the receptors was assessed by in vivo 32P labeling followed by immunoprecipitation with FLAG antibody and detection by SDS-PAGE and autoradiography. Lane 1, without increased B expression; lane 2, with increased B expression.

Increased expression of B regulates TGF- responsiveness. The lack of detectable effect of B on receptor phosphorylation does not preclude that B might regulate downstream TGF- signaling. We therefore tested the effect of B on two TGF- responses in HaCaT cells, a cell line which is highly sensitive to the antiproliferative effect of TGF- (16). In one set of assays, TGF--induced gene expression was assessed by measuring luciferase expression from the PAI-1 promoter (27) or the 3TP promoter (47) or by measuring PAI-1 protein expression (22) in stably transfected cells. TGF- greatly induced transcription from the 3TP promoter (Fig. 6A) or the PAI-1 promoter (data not shown). Increased B expression resulted in a small but reproducible decrease in the TGF- response without affecting the basal level of expression from the PAI-1 promoter (Fig. 6A). This decrease was also apparent in Mv1Lu epithelial cells (data not shown). Expression of the PAI-1 protein in HaCaT cells, stably transfected with a B expression plasmid, was, however, not decreased when compared to that of control transfected cells (Fig. 6A, inset). The discrepancy between these results may be due to measuring PAI-1 transcription versus protein levels.

View larger version (14K): [in this window] [in a new window]   FIG. 6.   Effect of B on TGF- responsiveness. (A) Effect of B on luciferase expression from the 3TP promoter. The B expression plasmid pRK7-B or the control plasmid pRK7 was cotransfected with p3TP-lux in HaCaT cells. Luciferase expression was measured in the absence or presence of TGF-. The inset shows that PAI-1 protein levels are not altered by B in stably transfected cell lines. 35S-labeled PAI-1 protein secreted from HaCaT cells, stably transfected with pRK7 (control) or pRK7-B in the absence () or presence (+) of 100 pM TGF-, was used. (B) B enhances the inhibition of luciferase activity by TGF- from the cyclin A promoter. HaCaT cells were cotransfected with the pCal2 luciferase reporter plasmid and the control plasmid pRK7 or the B expression plasmid pRK7-B. Luciferase expression from the cyclin A promoter, a measure of cell proliferation, was measured in the absence or presence of TGF-. the inset shows that B increases the inhibition of DNA synthesis by TGF- in stably transfected cells. DNA synthesis, measured by [3H]thymidine incorporation in HaCaT cells, stably transfected with pRK7 (control) or pRK7-B in the absence () or presence (+) of 100 pM TGF-, is shown. Data are presented relative to those of untreated, control transfected cells. Standard deviations were based on triplicate measurements.

View larger version (17K): [in this window] [in a new window]   FIG. 7.   B enhances the inhibition of cyclin A-luciferase activity independently from Smads. B increases the inhibition of cyclin A-luciferase activity comparably to Smad3 and -4. HaCaT (A) or Smad4-deficient SW480.7 (B) cells were cotransfected with pCal2 and pRK7 (control), pRK7-B, or pRK5 expression plasmids for Smad3 and -4. Luciferase expression from the cyclin A promoter was measured in the absence () or presence (+) of TGF-. B inhibition of cyclin A-luciferase activity occurs in the absence of Smad4 and is therefore Smad independent (B), whereas B and Smad3-Smad4 additively inhibit cyclin A-luciferase activity in these cells.

Effect of B on the growth inhibition response depends on functional TGF- receptors. The growth inhibition effect of B and its enhancement of the antiproliferative effect to exogenous TGF- in HaCaT cells (Fig. 6B) may be due to an effect of B, which is independent of TGF- receptor signaling, or may depend on autocrine receptor activation by endogenous TGF- produced by these cells (26). To distinguish between these possibilities, we analyzed the ability of B to inhibit the cyclin A response in mutant Mv1Lu cells, which lack functional receptors and therefore do not respond to TGF-, i.e., R1B cells lacking type I receptors and DR26 cells lacking functional type II receptors (29). As shown in Fig. 8A, B did not cause growth inhibition in receptor-deficient R1B or DR26 cells, indicating that the growth inhibition response to B requires functional TGF- receptors. This conclusion was confirmed by showing that reintroduction of functional type I receptors into R1B cells or type II receptors in DR26 cells restored the growth inhibition response of B (Fig. 8A). The sensitivity of the B-induced growth inhibition to functional receptors in the absence of exogenous TGF- is consistent with the ability of cells to respond to endogenous TGF- production and the consequent basal activation of the TGF- receptors (37). The dependence of the growth inhibition response of B on TGF- receptors was also illustrated by the effect of dominant-negative interference with TRI function. As shown in Fig. 8B, overexpression of a cytoplasmically truncated TRI, which inhibits receptor function (4, 14), inhibits the antiproliferative effect of B. Therefore, the enhancement of the growth inhibition response by B requires functional TGF- receptors.

View larger version (22K): [in this window] [in a new window]   View larger version (23K): [in this window] [in a new window]   FIG. 8.   Dependence of the growth inhibition effect of B on functional TGF- receptors. (A) The growth inhibition effect of B is TGF- receptor dependent. R1B cells, an Mv1Lu derivative lacking functional TRI, or DR26 cells, an Mv1Lu derivative lacking functional TRII, were cotransfected with the pCal2 cyclin A-luciferase plasmid and pRK7 expression plasmids for B, TRI/R4 (in R1B cells), or TRII (in DR26 cells). Luciferase expression from the cyclin A promoter was measured in the absence () or presence (+) of TGF-. (B) Dominant-negative inhibition of TR1/R4 activity blocks growth inhibition by B. Mv1Lu cells were cotransfected with the pCal2 cyclin A-luciferase plasmid and pRK7 expression plasmids for B or a cytoplasmically truncated form of TRI (DN R4) or both. Luciferase expression from the cyclin A promoter was measured in the absence or presence of TGF-. Luciferase values, normalized for transfection efficiency, are presented relative to those of untreated, control transfected cells. Standard deviations were based on triplicate measurements.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

In this study, we have shown that the WD-40 repeat-containing B subunit of PP2A can directly associate with the cytoplasmic domains of type I TGF- receptors. As a result of this interaction, B is phosphorylated by the type I receptor kinase. This physical interaction may provide a basis for the TGF- receptor-dependent growth inhibition effect of B, which complements the direct antiproliferative effect of TGF-, and may allow the type I receptor to regulate PP2A activity in a B-dependent manner. These data, together with the interaction of TRIP-1 with TGF- type II receptors, suggest that some WD-40 repeat proteins interact with serine/threonine kinase receptors.

Interaction of the B subunit of PP2A with TGF- receptors. The interaction of B, the WD-40 repeat subunit of PP2A, with the cytoplasmic domains of type I receptors was shown both in vitro and in vivo. Thus, purified B, with or without the AC core enzyme, associated directly with the cytoplasmic domains of type I receptors in vitro, and this association was confirmed in vivo. Furthermore, B also associated with type I receptors at the cell surface, as assessed by coimmunoprecipitation analyses of cell surface biotinylated receptors. Remarkably, B has a much lower affinity for the type II TGF- receptor, even though the cytoplasmic domains of TRII and the type I receptors have extensive sequence similarities. The association of B with the type I receptors parallels the association of TRIP-1, which, like B, has five WD-40 repeats, with TRII (8). WD-40 repeats may be involved in the interaction with the cytoplasmic domain of the receptor, as suggested by the interaction of both B and B with the type I receptor cytoplasmic domain, whereas B', which lacks WD-40 repeats, does not associate. Thus, TRIP-1 interacts with the type II receptor and not with the type I receptor, whereas B has a much higher affinity for the cytoplasmic domains of type I receptors than for the type II receptor.

The TGF- receptors can phosphorylate B. The interaction of the B subunit of PP2A with the type I receptor raises the possibility of functional interactions between receptor activation and the activity of PP2A. One aspect of this regulation is the phosphorylation of B by the type I receptor. Although phosphorylation of B was readily demonstrated in vitro, regulated phosphorylation of full-size B was not observed in vivo, possibly due to rapid dephosphorylation by PP2A. However, the use of a B mutant, which cannot associate with the AC core phosphatase, allowed us to visualize in vivo phosphorylation of B by type I receptors. Although the C subunit has previously been shown to be phosphorylated on tyrosine (6, 19), this is the first demonstration of phosphorylation of B.

The B subunit of PP2A regulates the TGF- response. Our results suggest that TGF- responsiveness can be regulated by B. Increased expression of B conferred a growth inhibition effect, which enhanced the antiproliferative response to TGF-. No effect of B on expression of TGF- or the TGF- receptors was observed (data not shown), whereas the effect on autocrine TGF- activation cannot be assessed. While the growth inhibition effect of B in the absence of added TGF- may have suggested a response independent of TGF- signaling, we found instead that this activity requires signaling by the receptors. This conclusion is based on our results with cells that lack functional receptors and with cells in which we overexpressed a dominant-negative mutant of the type I receptor (Fig. 8). Thus, the decreased cell proliferation induced by B in the absence of added TGF- is due to a sensitivity of the cells to endogenously produced TGF- and the consequent basal activation of the TGF- receptors (37). Finally, the dependence of the growth inhibition effect of B on functional TGF- receptors also supports the notion that TGF- receptors have the ability to regulate the activity of B.