Physical and functional interactions between type I transforming growth factor beta receptors and Balpha, a WD-40 repeat subunit of phosphatase 2A

Abstract

We have previously shown that a WD-40 repeat protein, TRIP-1, associates with the type II transforming growth factor beta (TGF-beta) receptor. In this report, we show that another WD-40 repeat protein, the Balpha subunit of protein phosphatase 2A, associates with the cytoplasmic domain of type I TGF-beta receptors. This association depends on the kinase activity of the type I receptor, is increased by coexpression of the type II receptor, which is known to phosphorylate and activate the type I receptor, and allows the type I receptor to phosphorylate Balpha. Furthermore, Balpha enhances the growth inhibition activity of TGF-beta in a receptor-dependent manner. Because Balpha has been characterized as a regulator of phosphatase 2A activity, our observations suggest possible functional interactions between the TGF-beta receptor complex and the regulation of protein phosphatase 2A.

FIG. 1

In vitro association of Bα with type I receptors. (A) Association of in vitro-translated Bα with GST-receptor fusion proteins. In vitro-translated ³⁵S-labeled Bα was incubated with glutathione-Sepharose-coupled GST (lane 2) or GST fused to the cytoplasmic domains of Tsk7L/R1 (lanes 3 and 5), TβRI/R4 (lane 4), or TβRII (lane 6). Bound proteins were eluted and separated by SDS-PAGE. Lane 1 shows 1/50 of the input ³⁵S-labeled Bα 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 Bα in the ABαC complex with the cytoplasmic domain of Tsk7L/R1. Purified ABαC 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-Bα antibody. The arrow points to the cross-linked complex of Bα with Tsk7L/R1 which is detected as an anti-Bα immunoreactive band. The arrowhead points to noncomplexed Bα. (E) Covalent cross-linking of Bβ in the ABβC complex with the cytoplasmic domain of Tsk7L/R1. Purified ABβC 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 Bβ with Tsk7L/R1 which is detected as the anti-Bβ immunoreactive band, and the arrowhead points to noncomplexed Bβ. (F) In vitro-translated B′ does not bind GST-receptor fusion proteins. In vitro-translated ³⁵S-labeled Bα (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 TβRI/R4 (lanes 3 and 6) as described for panel A.

FIG. 2

Interaction of Bα with the type I receptor cytoplasmic domain depends on its kinase activity and allows phosphorylation of Bα by the receptor kinase. (A) Bα interacts with kinase-active [ki(+)], not with kinase-inactive [ki(−)], Tsk7L/R1. Bα in the absence of the cytoplasmic domain of the receptor without (lane 1) or after (lane 2) chemical cross-linking is shown. Bα 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 Bα with Tsk7L/R1 which is detected as the anti-Bα immunoreactive band, and the arrowhead points to noncomplexed Bα. 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 Bα. The purified cytoplasmic domain of Tsk7L/R1 was incubated alone (lane 1), with Bα (lane 2), or with ABαC (lane 3) in a kinase reaction in the presence of [γ-³²P]ATP. Proteins were separated by SDS-PAGE, and ³²P-phosphorylated substrates were visualized by autoradiography. Besides Bα, a degradation product of Bα [Bα (degraded)] was also phosphorylated. The phosphorylated band above the Bα 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 ABαC (26), which was used in lane 3, with antibodies specific for A, Bα, or C, respectively.

FIG. 3

In vivo association of Bα with type I receptors. (A) In vivo association of Bα with type I receptors. Bα 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 TβRI/R4 (lanes 4 and 5) or the type II receptor TβRII (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 Bα was detected by immunoblotting with anti-Bα antibody. (B) TβRII coexpression enhances the association of Bα with TβRI. Bα was expressed alone (lane 1) or was coexpressed with the His₆-tagged cytoplasmic domain of TβRI/R4 (lanes 2 to 4) in transfected COS-1 cells, in the absence (lane 2) or presence (lanes 3 and 4) of TβRII (1 [lane 3] or 10 [lane 4] μg of transfected plasmid DNA). As in panel A, the proteins associated with TβRI purified on Co²⁺-Sepharose beads were separated by SDS-PAGE and associated Bα was detected by immunoblotting. (C) Interaction of Bα with full-size type I receptor at the cell surface. The full-size type I receptor Tsk7L/R1 (lanes 2 and 3) or TβRI/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 Bα in transfected cells. The corresponding cytoplasmically truncated Tsk7L/R1 (lane 1) or TβRI/R4 (lane 4) was also coexpressed with Bα. Cell surface proteins were labeled by surface biotinylation of intact cells, and immunoprecipitations were carried out by using anti-Bα 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.

FIG. 4

N-terminally truncated Bα is phosphorylated upon coexpression of TβRI and TβRII. Myc-epitope-tagged Bα, lacking amino acids 1 to 49, was coexpressed in the presence (+) or absence (−) of TβRI/R4 and TβRII (TβRI/II) in transfected COS cells. The in vivo phosphorylation of this deletion mutant of Bα 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 Bα mutant as assessed by anti-Myc Western blotting of anti-Myc immunoprecipitated protein. Left lanes, without TβRI/TβRII expression; right lanes, with TβRI/TβRII expression.

FIG. 5

Increased Bα expression does not alter the overall phosphorylation state of TGF-β receptors. FLAG-tagged type II receptors (TβRII) and type I receptor TβRI/R4 (R4) were coexpressed with Bα 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 Bα expression; lane 2, with increased Bα expression.

FIG. 6

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

FIG. 7

Bα enhances the inhibition of cyclin A-luciferase activity independently from Smads. Bα 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-Bα, or pRK5 expression plasmids for Smad3 and -4. Luciferase expression from the cyclin A promoter was measured in the absence (−) or presence (+) of TGF-β. Bα inhibition of cyclin A-luciferase activity occurs in the absence of Smad4 and is therefore Smad independent (B), whereas Bα and Smad3-Smad4 additively inhibit cyclin A-luciferase activity in these cells.

FIG. 8

Dependence of the growth inhibition effect of Bα on functional TGF-β receptors. (A) The growth inhibition effect of Bα is TGF-β receptor dependent. R1B cells, an Mv1Lu derivative lacking functional TβRI, or DR26 cells, an Mv1Lu derivative lacking functional TβRII, were cotransfected with the pCal2 cyclin A-luciferase plasmid and pRK7 expression plasmids for Bα, TβRI/R4 (in R1B cells), or TβRII (in DR26 cells). Luciferase expression from the cyclin A promoter was measured in the absence (−) or presence (+) of TGF-β. (B) Dominant-negative inhibition of TβR1/R4 activity blocks growth inhibition by Bα. Mv1Lu cells were cotransfected with the pCal2 cyclin A-luciferase plasmid and pRK7 expression plasmids for Bα or a cytoplasmically truncated form of TβRI (DN R4) or both. Luciferase expression from the cyclin A promoter was measured in the absence or presence of TGF-β. Luciferase values, normalized for transfection efficiency, are presented relative to those of untreated, control transfected cells. Standard deviations were based on triplicate measurements.

FIG. 8

Dependence of the growth inhibition effect of Bα on functional TGF-β receptors. (A) The growth inhibition effect of Bα is TGF-β receptor dependent. R1B cells, an Mv1Lu derivative lacking functional TβRI, or DR26 cells, an Mv1Lu derivative lacking functional TβRII, were cotransfected with the pCal2 cyclin A-luciferase plasmid and pRK7 expression plasmids for Bα, TβRI/R4 (in R1B cells), or TβRII (in DR26 cells). Luciferase expression from the cyclin A promoter was measured in the absence (−) or presence (+) of TGF-β. (B) Dominant-negative inhibition of TβR1/R4 activity blocks growth inhibition by Bα. Mv1Lu cells were cotransfected with the pCal2 cyclin A-luciferase plasmid and pRK7 expression plasmids for Bα or a cytoplasmically truncated form of TβRI (DN R4) or both. Luciferase expression from the cyclin A promoter was measured in the absence or presence of TGF-β. Luciferase values, normalized for transfection efficiency, are presented relative to those of untreated, control transfected cells. Standard deviations were based on triplicate measurements.