Distinct B subunits of PP2A regulate the NF‐κB signalling pathway through dephosphorylation of IKKβ, IκBα and RelA

PP2A is composed of a scaffolding subunit (A), a catalytic subunit (C) and a regulatory subunit (B) that is classified into four families including B, B′, B′′ and B′′′/striatin. Here, we found that a distinct PP2A complex regulates NF‐κB signalling by dephosphorylation of IKKβ, IκBα and RelA/p65. The PP2A core enzyme AC dimer and the holoenzyme AB′′′C trimer dephosphorylate IKKβ, IκBα and RelA, whereas the ABC trimer dephosphorylates IκBα but not IKKβ and RelA in cells. In contrast, AB′C and AB′′C trimers have little effect on dephosphorylation of these signalling proteins. These results suggest that different forms of PP2A regulate NF‐κB pathway signalling through multiple steps each in a different manner, thereby finely tuning NF‐κB‐ and IKKβ‐mediated cellular responses.

Edited by Kazuhiro Iwai PP2A is composed of a scaffolding subunit (A), a catalytic subunit (C) and a regulatory subunit (B) that is classified into four families including B, B 0 , B 00 and B 000 /striatin. Here, we found that a distinct PP2A complex regulates NF-jB signalling by dephosphorylation of IKKb, IjBa and RelA/p65. The PP2A core enzyme AC dimer and the holoenzyme AB 000 C trimer dephosphorylate IKKb, IjBa and RelA, whereas the ABC trimer dephosphorylates IjBa but not IKKb and RelA in cells. In contrast, AB 0 C and AB 00 C trimers have little effect on dephosphorylation of these signalling proteins. These results suggest that different forms of PP2A regulate NF-jB pathway signalling through multiple steps each in a different manner, thereby finely tuning NF-jB-and IKKb-mediated cellular responses.
Keywords: NF-kappa B; protein phosphatase; protein phosphatase 2 A (PP2A) Nuclear factor kappa B (NF-jB) is a critical transcription factor that regulates many cellular and organismal processes including immune and inflammatory responses, cellular growth and cell survival. The NF-jB signalling pathway is regulated by the phosphorylation of several proteins including inhibitor of kappa B (IjB) kinase b (IKKb), an inhibitory protein IjBa and an essential subunit of NF-jB RelA/p65 [1][2][3]. Binding of cytokines such as TNF-a to respective receptors leads to the conjugation of ubiquitin to several signalling components and recruits adaptor proteins and kinases [3][4][5][6]. These chains of reaction induce IKKb activation through phosphorylation of two critical serine residues, Ser177 and Ser181, at its activation loop [7,8]. IKKb in turn phosphorylates IjBa at the N-terminal serines, Ser32 and Ser36, which leads to ubiquitination at lysine residues Lys21 and Lys22 and IjBa degradation by the ubiquitin-proteasome system. These reactions result in the nuclear translocation of NF-jB and binding to its cognate jB sites in the promoters/enhancers of its target genes. IKKb also phosphorylates serine residues, Ser468 and Ser536, in the C-terminal transactivation domain of RelA and promotes gene expression through the association of RelA with a transcriptional coactivator, CREB-binding protein [9][10][11].
Among these phosphatases, PP2A is the most abundant, constituting approximately 1% of total cellular proteins [22][23][24]. PP2A is a heterotrimeric complex consisting of a scaffolding (A), regulatory (B) and catalytic (C) subunit. The A and C subunits each are comprised of two possible variants, a and b. The monomeric C subunit is unstable and requires binding to the A subunit to exist as a stable core enzyme AC dimer. The B subunit family consists of four classes including B (B55/ PR55), B 0 (B56/PR61), B 00 (PR48/PR72/PR130) and B 000 (PR93/PR110)/striatin (Strn). In turn, there are four isoforms of the B class including a, b, c and d, five of the B 0 class including a, b, c, d and e, three of the B 00 class including a, b and c, and three isoforms of the B 000 /Strn class including Strn, Strn3 and Strn4. These B subunits associate with the AC dimer to form the holoenzyme trimer. Crystal structure analysis revealed that the B subunits bind proximal to the C subunit active site in the holoenzyme trimer and determine the specificity for substrate proteins [25,26].
A previous RNAi screen revealed that PP2A plays an important role in the regulation of NF-jB signalling and identified the core enzyme subunits, Aa, Ab, Ca and Cb, as negative regulators of the NF-jB signalling pathway in mouse astrocytes [16]. However, this screen did not clarify whether the B subunit is involved in regulation of NF-jB activity, nor identify the specific B subunits regulating each NF-jB signalling step. It is plausible that, as cells usually express multiple isoforms of each B family protein, this failure was a result of complementation with other isoforms of the same family following the simple RNAi screen. In this study, we expressed B subunits together with A and C subunits in cells and investigated the role of these subunits on TNF-a-, IKKb-and RelA-induced NF-jB activation. This assay revealed that distinct B subunits regulate specific steps of NF-jB signalling. Dephosphorylation of IKKb and RelA is mediated by the AC dimer and AB 000 C trimer, whereas dephosphorylation of IjBa is mediated by the AC dimer, ABC trimer and AB 000 C trimer.

Materials and methods
Cell cultures, plasmids and transfection cDNA-encoding Aa, Ab, Ca, Cb, Ba, Bb, Bc, Bd, B 0 a, B 0 b, B 0 c, B 0 d, B 0 e, B 00 b, Strn and Strn3 subunits of PP2A were amplified from a human cDNA library by PCR. The cDNAs were inserted into pRK-HA and pRK-Flag expression vectors. Expression plasmids encoding IKKb, kinasenegative IKKb mutant (IKKbKN) and IjBa have been reported previously [27]. Constitutive active IKKbEE mutant of which phosphorylation sites Ser177/181 in the activation loop were exchanged to Glu, the ubiquitinationresistant IjBaRR mutant of which ubiquitination sites lysine residues Lys21/22 were exchanged to Arg, and the phosphatase-inactive CaD85N mutant of which Asp 85 was exchanged to Gln were generated using the KOD-Plus Mutagenesis kit (TOYOBO). Plasmids were transfected into GP2-293 cells in Opti-MEM (Invitrogen) using Lipofectamine Plus (Invitrogen) following the manufacturer's instructions. GP2-293 cells were obtained from Clontech. Cells were cultured in DMEM supplemented with 10% FBS containing 2 mM L-glutamine at 37°C.

Luciferase assay
Nuclear factor kappa B activity was estimated by Dual-Luciferase Reporter Assay System (Promega) using pNF-jB and pRL-TK Luciferase reporter plasmids. After transfection of 0.01 lg NF-jB reporter plasmids with or without 0.01 lg expression plasmids of various PP2A subunits, IKKb, IjBa and/or RelA into GP2-293 cells in collagencoated 96-well dishes, cells were incubated in the presence or absence of 50 ngÁmL À1 TNF-a for 18 h. After lysing cells with buffer from the assay system, luciferase activity was analysed following the manufacturer's instructions.

Results
The PP2A core enzyme downregulates NF-jB signalling PP2A is composed of three subunits scaffolding (A), regulatory (B) and catalytic (C) subunit (Fig. 1A). Cells were transfected with expression plasmids of a and b isoforms of the A and C subunits, and the expression of PP2A proteins were analysed by immunoblotting (Fig. 1B). Then, the effects of expression of these proteins on NF-jB activity were analysed by luciferase assay. Expression of the Ca and Cb subunits and of the C subunits together with Aa or Ab subunits suppressed TNF-a-induced NF-jB activation (Fig. 1C) and suppressed IKKb and constitutive active IKKbEE-induced NF-jB activation (Fig. 1D,E). In contrast, expression of the PP2A did not attenuate RelA-mediated NF-jB activity (Fig. 1F). These results suggest that PP2A suppresses NF-jB activity by suppressing IjBa and/or IKKb phosphorylation. Expression of inactive PP2A core enzyme consisting of the CaD85N mutant did not suppress TNF-a-, IKKband IKKbEE-induced NF-jB activation (Fig. 1G,H).

Distinct PP2A holoenzymes downregulate NF-jB signalling
To investigate the involvement of the PP2A in regulation of NF-jB signalling, we constructed expression plasmids encoding each subunit of the B, B 00 , B 00 and B 000 /Strn family ( Fig. 2A). Among isoforms of B 00 and B 000 /Strn families, we used expression plasmids encoding B 00 b for the B 00 family, along with Strn and Strn3 for the B 000 /Strn family, as B 00 a, B 00 c and B 000 /Strn4 hardly express in cells. Cells were transfected with NF-jB reporter plasmids and plasmids encoding Aa, Ca and each B subunit, and NF-jB activity was investigated following TNF-a treatment by luciferase assay. Expression of the AC holoenzyme, the ABC holoenzyme including A(Ba)C, A(Bb)C, A(Bc)C and A(Bd) C, and the AB 000 C holoenzyme including A(Strn)C and A(Strn3)C suppressed TNF-a-induced NF-jB activity, whereas the AB 0 C holoenzyme including A(B 0 a)C, A (B 0 b)C, A(B 0 c)C, A(B 0 d)C and A(B 0 e)C and the AB 00 C holoenzyme including A(B 00 b)C had little effect on TNF-a-induced NF-jB activation (Fig. 2B). Then, we investigated the effects of distinct B subunits on NF-jB signalling. NF-jB activity was analysed in cells transfected with plasmids encoding IKKb or IKKbEE together with Aa, Ca and each B subunit. A luciferase assay demonstrated that expression of AC, ABC and AB 000 C attenuated IKKb-and IKKbEE-induced NF-jB activity, whereas AB 0 C and AB 00 C had little effect on NF-jB activation (Fig. 2C,D). These results suggest that AC, ABC and AB 000 C suppress NF-jB activity downstream of IKKb. subunit, and phosphorylation of the activation loop was investigated by immunoblotting. Expression of the AC core enzyme markedly suppressed IKKb phosphorylation in a phosphatase activity-dependent manner (Fig. 3A). The AC core enzyme and the AB 000 C holoenzyme markedly suppressed IKKb phosphorylation, whereas three types of holoenzyme including ABC, AB 0 C and AB 00 C had little effect (Fig. 3B). These lines of evidence indicate that AC and AB 000 C attenuate TNF-a-induced NF-jB activity, at least in part, through dephosphorylation of IKKb.

AC and AB
To reveal the effects of each B subunit on the NF-jB/IjBa complex, we transfected cells with expression plasmids encoding IjBaRR, RelA and IKKbEE, Fig. 2. PP2A core enzyme AC and holoenzymes ABC and AB 000 C suppress TNF-a-and IKKb-induced NF-jB activity. (A) GP2-293 cells were transfected with plasmids encoding PP2A core enzyme subunits Aa and Ca together with subunits of B, B 0 , B 00 and B 000 /Strn families. Following incubation for 24 h, cells were lysed and expression of these subunits was analysed by immunoblotting. (B) Cells were transfected with NF-jB reporter plasmids and plasmids encoding PP2A subunits, and then stimulated with TNF-a for 18 h. NF-jB activity was estimated by luciferase assay. (C, D) Cells were transfected with NF-jB reporter plasmids and plasmids encoding PP2A subunits together with plasmids encoding IKKb (D) and IKKbEE (E). Cells were incubated for 18 h, and NF-jB activity was estimated by luciferase assay. *Statistically significance at P < 0.01. together with plasmids encoding Aa, Ca and each B subunit. Then, the effects of PP2A on IKKbEEmediated phosphorylation of IjBaRR and RelA were investigated by immunoblotting. Expression of the AC core enzyme suppressed phosphorylation of IjBaRR and RelA in a phosphatase activity-dependent manner (Fig. 3C). AC, ABC and AB 000 C reduced IjBa phosphorylation efficiently, whereas AB 0 C and AB 00 C had little effect on dephosphorylation of IjBaRR (Fig. 3D). In turn, AC and AB 000 C dephosphorylated RelA efficiently, whereas ABC, AB 0 C and AB 00 C had little effect on dephosphorylation of RelA. These results suggest that AC, ABC and AB 000 C attenuate NF-jB activity, at least in part, by dephosphorylation of IjBa.
Then, we investigated the effects of distinct B subunits on phosphorylation of endogenous NF-jB signalling proteins in TNF-a-stimulated cells. Cells were transfected with expression plasmids of PP2A, and then stimulated with TNF-a. AC and AB 000 C suppressed TNF-a-induced phosphorylation of IKKb and RelA, whereas ABC, AB 0 C and AB 00 C had little effect (Fig. 3E). AC, ABC and AB 000 C reduced TNF-ainduced IjBa phosphorylation, whereas AB 0 C and AB 00 C had little effect. These results suggest that functional features of B subunits have physiological significance in NF-jB signalling.
PP2A core enzyme and holoenzyme effectively dephosphorylate IKKb, IjBa and RelA in vitro Dephosphorylation of IKKb, IjBaRR and RelA was analysed by using purified PP2A enzyme in vitro. Expression plasmids encoding Flag-tagged Aa, HAtagged Ca and HA-tagged B subunits were transfected in cells, and PP2A complexes of various isoforms of B subunit family were purified from cells by using Flag-agarose beads (Fig. 4A). Phosphorylated IKKb was purified from cells transfected with Flag-IKKb expression plasmid (Fig. 4B). Following incubation of phosphorylated IKKb with various purified PP2A core enzyme and holoenzyme, dephosphorylation of IKKb was analysed by immunoblotting. This assay revealed that purified PP2A complexes of different B subunits effectively dephosphorylate IKKb, and these complexes showed similar levels of activity (Fig. 4C). Then, we purified phosphorylated complexes of IjBaRR and RelA from cells transfected with plasmids of Flag-RelA, HA-IjBaRR and IKKbEE by using Flag-agarose beads (Fig. 4D). Following incubation of phosphorylated IjBaRR and RelA protein complexes with various purified PP2A core enzyme and holoenzyme, dephosphorylation of IjBaRR and RelA was analysed by immunoblotting. Purified PP2A complexes of different B subunits effectively dephosphorylate IjBaRR and RelA (Fig. 4E). These results indicated that, although all PP2A complexes have an ability to dephosphorylate IKKb, IjBa and RelA in vitro, AC and AB 000 C preferentially dephosphorylate IKKb and RelA, and AC, ABC and AB 000 C preferentially dephosphorylate IjBa in vivo, suggesting that intracellular mechanisms may relate to the preference of AC, ABC and AB 000 C to dephosphorylate IKKb, IjBa and RelA.

Distinct B subunits regulate the binding of PP2A to the substrate proteins in vivo
To resolve the discrepancies in the dephosphorylation of IKKb and RelA between in vitro and in vitro, we investigated intracellular mechanisms of the preference of AC, ABC, AB 00 C and AB 000 C to dephosphorylate NF-jB signalling proteins. The effects of B subunits on the interaction between PP2A and IKKb were analysed by immunoprecipitation assay. Cells were transfected with plasmids encoding Flag-tagged CaD85N, HA-tagged Aa, HA-tagged B subunits and HA-tagged IKKb. PP2A complexes were immunoprecipitated by using Flag-agarose beads from cells, and the association of IKKb proteins to the PP2A complexes was analysed by immunoblotting (Fig. 5). This assay revealed that IKKb associates to PP2A, and B, B 0 and B″ subunits block the association. These results suggest that the preference of AC, ABC, AB 00 C and AB 000 C to dephosphorylate NF-jB signalling proteins is regulated by the binding features of B subunits to the substrate proteins in cells.

Discussion
Many studies have revealed important and complicated roles of PP2A in IKKb regulation. For example, it has been reported that PP2A suppresses IKKb activity in vitro and in vivo [7,15,16]. However, another contradictory study shows that PP2A forms a stable complex with IKKb and promotes its kinase activity in vivo [28]. We found here that AC and AB 000 C dephosphorylate IKKb in cells, whereas ABC, AB 0 C and AB 00 C do not. These results suggest the possibility that ABC, AB 0 C and AB 00 C complexes positively regulate IKKb by competing against AC and AB 000 C complexes. The data in this study may help to reconcile the seemingly contradictory observations that PP2A both downregulates and promotes IKKb activity. To confirm the specific regulation by distinct PP2A complexes, we tried to knockdown of multiple isoforms of each B family protein by RNAi. However, RNAi screen in GP2-293 cells did not clarify distinct functions of each isoform of B family protein, because expression levels of B and B‴ family proteins are very low in GP2-293 cells, and these cells express multiple isoforms of B 0 and B″ family proteins.
NF-jB signalling is not uniformly regulated by PP2A but rather is subjected to specific regulation by distinct PP2A complexes at multiple steps. In contrast to AC and AB 000 C, which dephosphorylate IKKb, IjBa and RelA, ABC dephosphorylates IjBa but not IKKb and RelA in cells. These results indicate that ABC suppresses NF-jB activity without inhibition of IKKb. A recent study revealed that IKKb not only  activates NF-jB through the phosphorylation of IjBa but also regulates many cellular functions by phosphorylating various proteins in an NF-jB-independent manner [29]. For example, IKKb regulates autophagy [30], mRNA stability [31], apoptosis [32], angiogenesis [33] and cellular vesicle trafficking [34] by phosphorylating various proteins that are unrelated to IjBa. Thus, ABC potentially suppresses NF-jB without inhibiting these NF-jB-independent functions of IKKb, thereby ensuring that PP2A regulates NF-jB activity and IKKb-mediated cellular responses independently.
Among the four classes of B subunit families, the B 000 /Strn family proteins only facilitate dephosphorylation of three proteins including IKKb, IjBa and RelA. Members of the B 000 /Strn family are evolutionarily conserved and have critical roles in biological processes such as development and cell growth [35,36]. B 000 /Strn family proteins form a large complex termed Striatininteracting phosphatase and kinase (STRIPAK) along with the germinal centre kinase family and many adaptor proteins, and recruit C subunits via A subunits of PP2A in the complexes. STRIPAK complexes have a critical role in protein dephosphorylation and act as important regulators of multiple vital signalling pathways, including the Hippo pathway, mitogen-activated protein kinases and cytoskeleton remodelling. Recent studies suggest that the dysregulation of STRIPAK complexes correlates with human diseases including cancer [35][36][37]. Regulation of NF-jB signalling pathway by AB‴C may, therefore, be involved in STRI-PAK complex-related biological processes and cancer.
PP2A is a confirmed tumour suppressor protein that is genetically altered or functionally inactivated in many cancers [22][23][24]. Its A and C subunits are reportedly inactivated through several mechanisms including somatic mutation, phosphorylation, methylation, and/ or increased expression of PP2A inhibitors, and inactivation of PP2A is linked to cancer progression. Expression of Ba, Bb and Bc is also decreased in many cancers owing to deletion, point mutation and DNA hypermethylation [38][39][40][41][42][43][44]. Furthermore, it has been revealed that IKKb and NF-jB signalling pathways are linked to inflammation and cancer [45]. Therefore, the regulation of IKKb and NF-jB signalling by distinct PP2A complexes may be involved in multiple human diseases including cancer, and thus might serve as specific and novel targets for disease therapy.