Alpha4 is a ubiquitin-binding protein that regulates protein serine/threonine phosphatase 2A ubiquitination

Abstract

Multiple regulatory mechanisms control the activity of the protein serine/threonine phosphatase 2A catalytic subunit (PP2Ac), including post-translational modifications and its association with regulatory subunits and interacting proteins. Alpha4 is a PP2Ac-interacting protein that is hypothesized to play a role in PP2Ac ubiquitination via its interaction with the E3 ubiquitin ligase Mid1. In this report, we show that alpha4 serves as a necessary adaptor protein that provides a binding platform for both PP2Ac and Mid1. We also identify a novel ubiquitin-interacting motif (UIM) within alpha4 (amino acid residues 46-60) and analyze the interaction between alpha4 and ubiquitin using NMR. Consistent with other UIM-containing proteins, alpha4 is monoubiquitinated. Interestingly, deletion of the UIM within alpha4 enhances its association with polyubiquitinated proteins. Lastly, we demonstrate that addition of wild-type alpha4 but not an alpha4 UIM deletion mutant suppresses PP2Ac polyubiquitination. Thus, the polyubiquitination of PP2Ac is inhibited by the UIM within alpha4. These findings reveal direct regulation of PP2Ac polyubiquitination by a novel UIM within the adaptor protein alpha4.

Fig. 1. Alpha4 is an adaptor protein linking Mid1 and PP2Ac

A, HEK293 cells were transfected with myc-Mid1, HA-PP2Ac, Flag-alpha4, or the indicated combinations of these constructs. Myc immune complexes (Myc IPs) were isolated from cell lysates and analyzed by SDS-PAGE and immunoblotting using myc, Flag, and HA antibodies to detect the epitope-tagged forms of Mid1, alpha4, and PP2Ac, respectively. Aliquots of the cell lysates were analyzed in the same manner. B, HEK293 cells were co-transfected with HA-ubiquitin and the indicated construct(s). Flag immune complexes (Flag IPs) were isolated from cell lysates and analyzed by SDS-PAGE and immunoblotting using myc, Flag, and PP2Ac antibodies to detect the epitope-tagged forms of Mid1 and alpha4, as well as endogenous PP2Ac. The data are representative of at least three separate experiments.

Fig. 2. Alpha4 contains a ubiquitin-interacting motif (UIM) and directly binds to ubiquitin

A, Helical wheel representation of the UIM within alpha4 (amino acids 46-59). Circles denote hydrophilic residues, diamonds denote hydrophobic residues, triangles denote acidic residues, and pentagons denote basic residues. Hydrophobic residues are green; the level of green decreases in scale with hydrophobicity. Hydrophilic residues are red; the level of red decreases in scale with hydrophilicity. Charged residues are light blue. B, Schematic representation of the UIM (46-60), PP2Ac-binding (98-202), and Mid1-binding (220-290) domains of alpha4. C, Binding of alpha4 to ubiquitin perturbs specific residues in the ubiquitin I44/UIM interaction interface. A region of the ¹H-¹⁵N HSQC spectra of Ub in the absence (black) and presence (red) of 12 molar equivalents of alpha4 demonstrate the selective line broadening and intensity loss observed upon formation of the Ub•alpha4 complex. The NMR data are representative of two independent experiments with five titration points each.

Fig. 3. Alpha4 is targeted for monoubiquitination

HEK293 cells were transfected with HA-ubiquitin, Flag-alpha4, or both HA-ubiquitin and Flag-alpha4. Immunoprecipitations were performed from the cell lysates using Flag M2 agarose (Flag IPs) or HA-agarose beads (HA IPs). Bound proteins were analyzed by SDS-PAGE and immunoblotting with HA and Flag antibodies. The asterisk denotes the migration of monoubiquitinated alpha4. Lysates were analyzed in the same manner (bottom panel). The migration of SDS-PAGE standards is shown on the left. The data are representative of at least three separate experiments.

Fig. 4. Deletion of the UIM within alpha4 results in enhanced binding to polyubiquitinated proteins

HEK293 cells were transfected with HA-ubiquitin, wild-type Flag-alpha4 (WT), or the combination of the indicated Flag-alpha4 protein (WT,Δ51-53, and Δ46-60) and HA-ubiquitin. Immunoprecipitations were performed from the cell lysates using Flag M2 agarose beads (Flag IPs). Bound proteins were analyzed by SDS-PAGE and immunoblotting using HA and Flag antibodies. Aliquots of the cell lysates were analyzed in the same manner. The migration of SDS-PAGE standards is shown on the left. The data are representative of at least three separate experiments.

Fig. 5. The UIM within alpha4 regulates PP2Ac ubiquitination

A, In vitro ubiquitination reactions were carried out using purified PP2Ac as a substrate in the absence (−) or presence (+) of Flag-alpha4 or Flag-alpha4 UIM deletion mutant Δ46-60. Reaction mixtures were analyzed by SDS-PAGE and immunoblotting with antibodies recognizing PP2Ac (top panel) and alpha4 (bottom panel). The ~35 kDa protein detected in reaction mixtures lacking purified PP2Ac (lane 2) represents PP2Ac present in the ubiquitin-protein conjugation kit; the weak PP2Ac polyubiquitination signal detected in this lane is defined as background PP2Ac ubiquitination. B, Immunoprecipitations were performed from ubiquitination reactions in A using Flag M2 agarose (Flag IPs), and analyzed by SDS-PAGE and immunoblotting with PP2Ac and alpha4 antibodies. The migration of SDS-PAGE standards is shown on the left. C, Higher intensity scan of top portion of the blot shown in B. The data are representative of at least three separate experiments.