The E3 ubiquitin ligase- and protein phosphatase 2A (PP2A)-binding domains of the Alpha4 protein are both required for Alpha4 to inhibit PP2A degradation

Abstract

Protein phosphatase 2A (PP2A) is regulated through a variety of mechanisms, including post-translational modifications and association with regulatory proteins. Alpha4 is one such regulatory protein that binds the PP2A catalytic subunit (PP2Ac) and protects it from polyubiquitination and degradation. Alpha4 is a multidomain protein with a C-terminal domain that binds Mid1, a putative E3 ubiquitin ligase, and an N-terminal domain containing the PP2Ac-binding site. In this work, we present the structure of the N-terminal domain of mammalian Alpha4 determined by x-ray crystallography and use double electron-electron resonance spectroscopy to show that it is a flexible tetratricopeptide repeat-like protein. Structurally, Alpha4 differs from its yeast homolog, Tap42, in two important ways: 1) the position of the helix containing the PP2Ac-binding residues is in a more open conformation, showing flexibility in this region; and 2) Alpha4 contains a ubiquitin-interacting motif. The effects of wild-type and mutant Alpha4 on PP2Ac ubiquitination and stability were examined in mammalian cells by performing tandem ubiquitin-binding entity precipitations and cycloheximide chase experiments. Our results reveal that both the C-terminal Mid1-binding domain and the PP2Ac-binding determinants are required for Alpha4-mediated protection of PP2Ac from polyubiquitination and degradation.

FIGURE 1.

Structure of Alpha4ΔC. A, ribbon diagram of Alpha4ΔC, with residues important for PP2Ac binding shown in orange and the consensus UIM shown in yellow. B, comparison of the Alpha4ΔC structure (blue) with the Tap42ΔC structures (cyan and magenta) showing the variable positions of the extended helix (residues 147–182). PyMOL was used to depict all molecular structures (48). MolA and MolB, molecules A and B, respectively.

FIGURE 2.

Distances between the spin label pair L206C/S154C computed via DEER/pulsed EPR studies. A, ribbon diagram showing the locations of spin labels L206C and S154C (green) and the distance between β-carbons. The UIM is shown in yellow, and PP2Ac-binding residues are shown in orange. B, distance distribution profiles corresponding to the best fit (shown in supplemental Fig. 1, A and B), showing a major distance distribution of ∼31 Å compared with ∼25 Å in the crystal structure.

FIGURE 3.

Comparison of Alpha4ΔC and TPR proteins. A, topology diagrams of TPR (upper) and Alpha4ΔC (lower) showing the altered topology of the final helices. The part represented in gray is based on the crystal structure of Tap42ΔC, as these residues are not observed in the crystal structure of Alpha4ΔC. The diagrams were created in TOPDRAW (49). B, structures and surface representations of TPR (upper) and Alpha4ΔC (lower) showing the configuration of helices and the formation of the concave and convex surfaces (with the outline of concavity denoted by the dotted line). C, superposition of Alpha4ΔC (colored blue, yellow, and orange as in Fig. 1) with the SycD TPR domain (pink; Protein Data Bank code 2VGY) and 14-3-3 (green; code 3EFZ) reveals similar tertiary structures but indicates that the concave face of Alpha4 is more closed than the canonical TPR and 14-3-3 proteins. The helices shown in cyan represent helices from Tap42ΔC that differ significantly in position from those in Alpha4ΔC.

FIGURE 4.

Alpha4ΔC binds PP2A but fails to bind Mid1. A, HEK293FT whole cell lysate was incubated with cobalt-nitrilotriacetic acid resin in the absence (−) or presence (+) of either Alpha4ΔC or Alpha4ΔC_AA (mutation of the PP2Ac-binding residues). Bound proteins were eluted with 200 mm imidazole, and the eluate was analyzed by SDS-PAGE and immunoblotting with antibodies recognizing PP2Ac (upper panel) and the His₆ tag (lower panel). Data are representative of three independent experiments. WB, Western blot. B, HEK293FT cells were transfected with HA₃-PP2Ac, FLAG-Alpha4, FLAG-Alpha4ΔC, Myc-Mid1, or a combination of the constructs. FLAG immune complexes (Flag IPs) were isolated from the cells and subjected to SDS-PAGE and Western analysis using anti-Myc, anti-PP2Ac, and anti-Alpha4 antibodies.

FIGURE 5.

Both the Mid1-binding domain and PP2Ac binding are essential for Alpha4 inhibition of PP2Ac polyubiquitination. HEK293FT cells were transfected with HA₃-PP2Ac and empty vector, full-length WT FLAG-Alpha4, FLAG-Alpha4ΔC, or FLAG-Alpha4_ED. At 48 h post-transfection, cells were treated with 25 μm proteasome inhibitor (MG132) for 4 h at 37 °C prior to lysis. Total polyubiquitinated proteins were isolated from the cell lysates using TUBE2-agarose beads. Protein expression and the polyubiquitination state of ectopic PP2Ac were analyzed via immunoblotting using anti-PP2Ac, anti-Alpha4, and anti-ubiquitin antibodies.

FIGURE 6.

The Mid1-binding domain of Alpha4 is essential to protect PP2Ac from degradation. HEK293FT cells were transfected with HA₃-PP2Ac alone or with HA₃-PP2Ac and FLAG-Alpha4, FLAG-Alpha4ΔC, or FLAG-Alpha4_ED. Cells were treated with 50 μm cycloheximide (CHX) 48 h post-transfection and then lysed at the indicated time points post-treatment. The lysates were subjected to Western analysis using antibodies recognizing Alpha4, PP2Ac, and HSP90.