IL-17A's role in exacerbating radiation-induced lung injury: Autophagy impairment via the PP2A-mTOR pathway.

OBJECTIVE: Radiation-induced lung injury (RILI) is a serious complication of radiotherapy, and the role of IL-17A in this process is not well understood. While IL-17A has been shown to modulate autophagy, conflicting reports exist regarding its activation or inhibition of autophagy. This study investigates the role of IL-17A in RILI and its effects on autophagy via the PP2A-mTOR pathway, with a focus on the PP2A B56α subunit. METHODS: C57BL/6J mice and human lung epithelial cells (BEAS-2B) were exposed to radiation with or without recombinant IL-17A. Autophagy markers were analyzed using Western blotting, immunofluorescence, and autophagy flux assays. PP2A activity, specifically the B56α subunit, was measured. A PP2A agonist (DT-061) was used to verify its role in reversing IL-17A-mediated autophagy inhibition. RESULTS: IL-17A inhibited autophagy in lung epithelial cells exposed to radiation by suppressing PP2A activity, particularly through downregulation of the B56α subunit, leading to mTOR activation and reduced autophagosome formation. Treatment with DT-061 restored autophagic activity and improved cell viability. These findings align with reports suggesting that IL-17A inhibits autophagy in certain contexts, while other studies have shown opposing effects. CONCLUSION: IL-17A inhibits autophagy in RILI through the PP2A B56α-mTOR pathway, exacerbating lung damage. Further research is needed to clarify the role of IL-17A in different cell types and conditions. Targeting the IL-17A-PP2A B56α-mTOR axis may offer new therapeutic strategies for RILI management.

The cellular protein phosphatase 2A is a crucial host factor for Marburg virus transcription.

Little is known regarding the molecular mechanisms that highly pathogenic Marburg virus (MARV) utilizes to transcribe and replicate its genome. Previous studies assumed that dephosphorylation of the filoviral transcription factor VP30 supports transcription, while phosphorylated VP30 reduces transcription. Here, we focused on the role of the host protein phosphatase 2A (PP2A) for VP30 dephosphorylation and promotion of viral transcription. We could show that MARV NP interacts with the subunit B56 of PP2A, as previously shown for the Ebola virus, and that this interaction is important for MARV transcription activity. Inhibition of the interaction between PP2A and NP either by mutating the B56 binding motif encoded on NP, or the use of a PP2A inhibitor, induced VP30 hyperphosphorylation, and as a consequence a decrease of MARV transcription as well as viral growth. These results suggest that NP plays a key role in the dephosphorylation of VP30 by recruiting PP2A. Generation of recombinant (rec) MARV lacking the PP2A-B56 interaction motif on NP was not possible suggesting an essential role of PP2A-mediated VP30 dephosphorylation for the MARV replication cycle. Likewise, we were not able to generate recMARV containing VP30 phosphomimetic mutants indicating that dynamic cycles of VP30 de- and rephosphorylation are a prerequisite for an efficient viral life cycle. As the specific binding motifs of PP2A-B56 and VP30 within NP are highly conserved among the filoviral family, our data suggest a conserved mechanism for filovirus VP30 dephosphorylation by PP2A, revealing the host factor PP2A as a promising target for pan-filoviral therapies. IMPORTANCE: Our study elucidates the crucial role of host protein phosphatase 2A (PP2A) in Marburg virus (MARV) transcription. The regulatory subunit B56 of PP2A facilitates VP30 dephosphorylation, and hence transcription activation, via binding to NP. Our results, together with previous data, reveal a conserved mechanism of filovirus VP30 dephosphorylation by host factor PP2A at the NP interface and provide novel insights into potential pan-filovirus therapies.

Emerging Roles of B56 Phosphorylation and Binding Motif in PP2A-B56 Holoenzyme Biological Function.

Protein serine/threonine phosphatase 2A (PP2A) regulates diverse cellular processes via the formation of ~100 heterotrimeric holoenzymes. However, a scarcity of knowledge on substrate recognition by various PP2A holoenzymes has greatly prevented the deciphering of PP2A function in phosphorylation-mediated signaling in eukaryotes. The review summarized the contribution of B56 phosphorylation to PP2A-B56 function and proposed strategies for intervening B56 phosphorylation to treat diseases associated with PP2A-B56 dysfunction; it especially analyzed recent advancements in LxxIxEx B56-binding motifs that provide the molecular details of PP2A-B56 binding specificity and, on this basis, explored the emerging role of PP2A-B56 in the mitosis process, virus attack, and cancer development through LxxIxE motif-mediated PP2A-B56 targeting. This review provides theoretical support for discriminatingly targeting specific PP2A holoenzymes to guide PP2A activity against specific pathogenic drivers.

A bifunctional kinase-phosphatase module balances mitotic checkpoint strength and kinetochore-microtubule attachment stability.

Two major mechanisms safeguard genome stability during mitosis: the mitotic checkpoint delays mitosis until all chromosomes have attached to microtubules, and the kinetochore-microtubule error-correction pathway keeps this attachment process free from errors. We demonstrate here that the optimal strength and dynamics of these processes are set by a kinase-phosphatase pair (PLK1-PP2A) that engage in negative feedback from adjacent phospho-binding motifs on the BUB complex. Uncoupling this feedback to skew the balance towards PLK1 produces a strong checkpoint, hypostable microtubule attachments and mitotic delays. Conversely, skewing the balance towards PP2A causes a weak checkpoint, hyperstable microtubule attachments and chromosome segregation errors. These phenotypes are associated with altered BUB complex recruitment to KNL1-MELT motifs, implicating PLK1-PP2A in controlling auto-amplification of MELT phosphorylation. In support, KNL1-BUB disassembly becomes contingent on PLK1 inhibition when KNL1 is engineered to contain excess MELT motifs. This elevates BUB-PLK1/PP2A complex levels on metaphase kinetochores, stabilises kinetochore-microtubule attachments, induces chromosome segregation defects and prevents KNL1-BUB disassembly at anaphase. Together, these data demonstrate how a bifunctional PLK1/PP2A module has evolved together with the MELT motifs to optimise BUB complex dynamics and ensure accurate chromosome segregation.

Spatial separation of phosphatase and kinase activity within the Bub complex is required for proper mitosis.

The Bub1 and BubR1 kinetochore proteins support proper chromosome segregation and mitotic checkpoint activity. Bub1 and BubR1 are paralogs with Bub1 being a kinase, while BubR1 localizes the PP2A-B56 protein phosphatase to kinetochores in humans. Whether this spatial separation of kinase and phosphatase activity is important is unclear as some organisms integrate both activities into one Bub protein. Here, we engineer human Bub1 and BubR1 proteins integrating kinase and phosphatase activities into one protein and show that these do not support normal mitotic progression. A Bub1-PP2A-B56 complex can support chromosome alignment but results in impairment of the checkpoint due to dephosphorylation of the Mad1 binding site in Bub1. Furthermore, a chimeric BubR1 protein containing the Bub1 kinase domain induces delocalized H2ApT120 phosphorylation, resulting in the reduction of centromeric hSgo2 and chromosome segregation errors. Collectively, these results argue that the spatial separation of kinase and phosphatase activities within the Bub complex is required for balancing its functions in the checkpoint and chromosome alignment.

MPS1 localizes to end-on microtubule-attached kinetochores to promote microtubule release.

In eukaryotes, the spindle assembly checkpoint protects genome stability in mitosis by preventing chromosome segregation until incorrect microtubule-kinetochore attachment geometries have been eliminated and chromosome biorientation has been completed. These error correction and checkpoint processes are linked by the conserved Aurora B and MPS1 Ser/Thr kinases.1,2 MPS1-dependent checkpoint signaling is believed to be initiated by kinetochores without end-on microtubule attachments,3,4 including those generated by Aurora B-mediated error correction. The current model posits that MPS1 competes with microtubules for binding sites at the kinetochore.3,4 MPS1 is thought to first recognize kinetochores not blocked by microtubules and then initiate checkpoint signaling. However, MPS1 is also required for chromosome biorientation and correction of microtubule-kinetochore attachment errors.5,6,7,8,9 This latter function, which must require direct interaction with microtubule-attached kinetochores, is not readily explained within the constraints of the current model. Here, we show that MPS1 transiently localizes to end-on attached kinetochores and that this recruitment depends on the relative activities of Aurora B and its counteracting phosphatase PP2A-B56 rather than microtubule-attachment state per se. MPS1 autophosphorylation also regulates MPS1 kinetochore levels but does not determine the response to microtubule attachment. At end-on attached kinetochores, MPS1 actively promotes microtubule release together with Aurora B. Furthermore, in live cells, MPS1 is detected at attached kinetochores before the removal of microtubules. During chromosome alignment, MPS1, therefore, coordinates both the resolution of incorrect microtubule-kinetochore attachments and the initiation of spindle checkpoint signaling.

Protein Phosphatase 2A with B' specificity subunits regulates the Hippo-Yorkie signaling axis in the Drosophila eye disc.

Hippo-Yorkie (Hpo-Yki) signaling is central to diverse developmental processes. Although its redeployment has been amply demonstrated, its context-specific regulation remains poorly understood. The Drosophila eye disc is a continuous epithelium folded into two layers, the peripodial epithelium (PE) and the retinal progenitor epithelium. Here, Yki acts in the PE, first to promote PE identity by suppressing retina fate, and subsequently to maintain proper disc morphology. In the latter process, loss of Yki results in the displacement of a portion of the differentiating retinal epithelium onto the PE side. We show that Protein Phosphatase 2A (PP2A) complexes comprising different substrate-specificity B-type subunits govern the Hpo-Yki axis in this context. These include holoenzymes containing the B‴ subunit Cka and those containing the B' subunits Wdb or Wrd. Whereas PP2A(Cka), as part of the STRIPAK complex, is known to regulate Hpo directly, PP2A(Wdb) acts genetically upstream of the antagonistic activities of the Hpo regulators Sav and Rassf. These in vivo data provide the first evidence of PP2A(B') heterotrimer function in Hpo pathway regulation and reveal pathway diversification at distinct developmental times in the same tissue.

Uncoupling of Mitosis and Cytokinesis Upon a Prolonged Arrest in Metaphase Is Influenced by Protein Phosphatases and Mitotic Transcription in Fission Yeast.

Depletion of the Anaphase-Promoting Complex/Cyclosome (APC/C) activator Cdc20 arrests cells in metaphase with high levels of the mitotic cyclin (Cyclin B) and the Separase inhibitor Securin. In mammalian cells this arrest has been exploited for the treatment of cancer with drugs that engage the spindle assembly checkpoint and, recently, with chemical inhibitors of the APC/C. While most cells arrested in mitosis for prolonged periods undergo apoptosis, others skip cytokinesis and enter G1 with unsegregated chromosomes. This process, known as mitotic slippage, generates aneuploidy and increases genomic instability in the cancer cell. Here, we analyze the behavior of fission yeast cells arrested in mitosis through the transcriptional silencing of the Cdc20 homolog slp1. While depletion of slp1 readily halts cells in metaphase, this arrest is only transient and a majority of cells eventually undergo cytokinesis and show steady mitotic dephosphorylation. Notably, this occurs in the absence of Cyclin B (Cdc13) degradation. We investigate the involvement of phosphatase activity in these events and demonstrate that PP2A-B55Pab1 is required to prevent septation and, during the arrest, its CDK-mediated inhibition facilitates the induction of cytokinesis. In contrast, deletion of PP2A-B56Par1 completely abrogates septation. We show that this effect is partly due to this mutant entering mitosis with reduced CDK activity. Interestingly, both PP2A-B55Pab1 and PP2A-B56Par1, as well as Clp1 (the homolog of the budding yeast mitotic phosphatase Cdc14) are required for the dephosphorylation of mitotic substrates during the escape. Finally, we show that the mitotic transcriptional wave controlled by the RFX transcription factor Sak1 facilitates the induction of cytokinesis and also requires the activity of PP2A-B56Par1 in a mechanism independent of CDK.

Impaired myocellular Ca2+ cycling in protein phosphatase PP2A-B56α KO mice is normalized by ß-adrenergic stimulation.

The activity of protein phosphatase 2A (PP2A) is determined by the expression and localization of the regulatory B-subunits. PP2A-B56α is the dominant isoform of the B'-family in the heart. Its role in regulating the cardiac response to ß-adrenergic stimulation is not yet fully understood. We therefore generated mice deficient in B56α to test the functional cardiac effects in response to catecholamine administration versus corresponding WT mice. We found the decrease in basal PP2A activity in hearts of KO mice was accompanied by a counter-regulatory increase in the expression of B' subunits (ß and γ) and higher phosphorylation of sarcoplasmic reticulum Ca2+ regulatory and myofilament proteins. The higher phosphorylation levels were associated with enhanced intraventricular pressure and relaxation in catheterized KO mice. In contrast, at the cellular level, we detected depressed Ca2+ transient and sarcomere shortening parameters in KO mice at basal conditions. Consistently, the peak amplitude of the L-type Ca2+ current was reduced and the inactivation kinetics of ICaL were prolonged in KO cardiomyocytes. However, we show ß-adrenergic stimulation resulted in a comparable peak amplitude of Ca2+ transients and myocellular contraction between KO and WT cardiomyocytes. Therefore, we propose higher isoprenaline-induced Ca2+ spark frequencies might facilitate the normalized Ca2+ signaling in KO cardiomyocytes. In addition, the application of isoprenaline was associated with unchanged L-type Ca2+ current parameters between both groups. Our data suggest an important influence of PP2A-B56α on the regulation of Ca2+ signaling and contractility in response to ß-adrenergic stimulation in the myocardium.

Molecular dissection on inhibition of Ras-induced cellular senescence by small t antigen of SV40.

Simian virus 40 (SV40) is a potentially oncogenic virus of monkey origin. Transmission, prevalence, and pathogenicity rates of SV40 are unclear, but infection can occur in humans, for example individuals with high contact with rhesus macaques and individuals that received contaminated early batches of polio vaccines in 1950-1963. In addition, several human polyomaviruses, proven carcinogenic, are also highly common in global populations. Cellular senescence is a major mechanism of cancer prevention in vivo. Hyperactivation of Ras usually induces cellular senescence rather than cell transformation. Previous studies suggest small t antigen (ST) of SV40 may interfere with cellular senescence induced by Ras. In the current study, ST was demonstrated to inhibit Ras-induced cellular senescence (RIS) and accumulation of DNA damage in Ras-activated cells. In addition, ST suppressed the signal transmission from BRaf to MEK and thus blocked the downstream transmission of the activated Ras signal. B56γ knockdown mimicked the inhibitory effects of ST overexpression on RIS. Furthermore, KSR1 knockdown inhibited Ras activation and the subsequent cellular senescence. Further mechanism studies indicated that the phosphorylation level of KSR1 rather than the levels of the total protein regulates the activation of Ras signaling pathway. In sum, ST inhibits the continuous hyperactivation of Ras signals by interfering with the normal functions of PP2A-B56γ of dephosphorylating KSR1, thus inhibiting the occurrence of cellular senescence. Although the roles of SV40 in human carcinogenesis are controversial so far, our study has shown that ST of polyomaviruses has tumorigenic potential by inhibiting oncogene-induced senescence (OIS) as a proof of concept.

A highly conserved pocket on PP2A-B56 is required for hSgo1 binding and cohesion protection during mitosis.

The shugoshin proteins are universal protectors of centromeric cohesin during mitosis and meiosis. The binding of human hSgo1 to the PP2A-B56 phosphatase through a coiled-coil (CC) region mediates cohesion protection during mitosis. Here we undertook a structure function analysis of the PP2A-B56-hSgo1 complex, revealing unanticipated aspects of complex formation and function. We establish that a highly conserved pocket on the B56 regulatory subunit is required for hSgo1 binding and cohesion protection during mitosis in human somatic cells. Consistent with this, we show that hSgo1 blocks the binding of PP2A-B56 substrates containing a canonical B56 binding motif. We find that PP2A-B56 bound to hSgo1 dephosphorylates Cdk1 sites on hSgo1 itself to modulate cohesin interactions. Collectively our work provides important insight into cohesion protection during mitosis.

A dynamic charge-charge interaction modulates PP2A:B56 substrate recruitment.

The recruitment of substrates by the ser/thr protein phosphatase 2A (PP2A) is poorly understood, limiting our understanding of PP2A-regulated signaling. Recently, the first PP2A:B56 consensus binding motif, LxxIxE, was identified. However, most validated LxxIxE motifs bind PP2A:B56 with micromolar affinities, suggesting that additional motifs exist to enhance PP2A:B56 binding. Here, we report the requirement of a positively charged motif in a subset of PP2A:B56 interactors, including KIF4A, to facilitate B56 binding via dynamic, electrostatic interactions. Using molecular and cellular experiments, we show that a conserved, negatively charged groove on B56 mediates dynamic binding. We also discovered that this positively charged motif, in addition to facilitating KIF4A dephosphorylation, is essential for condensin I binding, a function distinct and exclusive from PP2A-B56 binding. Together, these results reveal how dynamic, charge-charge interactions fine-tune the interactions mediated by specific motifs, providing a new framework for understanding how PP2A regulation drives cellular signaling.

An Attachment-Independent Biochemical Timer of the Spindle Assembly Checkpoint.

The spindle assembly checkpoint (SAC) generates a diffusible protein complex that prevents anaphase until all chromosomes are properly attached to spindle microtubules. A key step in SAC initiation is the recruitment of MAD1 to kinetochores, which is generally thought to be governed by the microtubule-kinetochore (MT-KT) attachment status. However, we demonstrate that the recruitment of MAD1 via BUB1, a conserved kinetochore receptor, is not affected by MT-KT interactions in human cells. Instead, BUB1:MAD1 interaction depends on BUB1 phosphorylation, which is controlled by a biochemical timer that integrates counteracting kinase and phosphatase effects on BUB1 into a pulse-generating incoherent feedforward loop. We propose that this attachment-independent timer serves to rapidly activate the SAC at mitotic entry, before the attachment-sensing MAD1 receptors have become fully operational. The BUB1-centered timer is largely impervious to conventional anti-mitotic drugs, and it is, therefore, a promising therapeutic target to induce cell death through permanent SAC activation.

PP2A-B56γ is required for an efficient spindle assembly checkpoint.

The Spindle Assembly Checkpoint (SAC) is part of a complex feedback system designed to ensure that cells do not proceed through mitosis unless all chromosomal kinetochores have attached to spindle microtubules. The formation of the kinetochore complex and the implementation of the SAC are regulated by multiple kinases and phosphatases. BubR1 is a phosphoprotein that is part of the Cdc20 containing mitotic checkpoint complex that inhibits the APC/C so that Cyclin B1 and Securin are not degraded, thus preventing cells going into anaphase. In this study, we found that PP2A in association with its B56γ regulatory subunit, are needed for the stability of BubR1 during nocodazole induced cell cycle arrest. In primary cells that lack B56γ, BubR1 is prematurely degraded and the cells proceed through mitosis. The reduced SAC efficiency results in cells with abnormal chromosomal segregation, a hallmark of transformed cells. Previous studies on PP2A's role in the SAC and kinetochore formation were done using siRNAs to all 5 of the B56 family members. In our study we show that inactivation of only the PP2A-B56γ subunit can affect the efficiency of the SAC. We also provide data that show the intracellular locations of the B56 subunits varies between family members, which is consistent with the hypothesis that they are not completely functionally redundant.

B56δ subunit of protein phosphatase 2A decreases phosphorylation of endothelial nitric oxide synthase at serine 116: Mechanism underlying aphidicolin-stimulated NO production.

DNA damage is significant in endothelial cells (EC), particularly in anticancer chemotherapy. Here, we explored whether and how aphidicolin, a DNA-damaging chemical with a promising anticancer activity, alters NO production in bovine aortic endothelial cells (BAEC). In addition to increasing eNOS-Ser1179 phosphorylation, aphidicolin decreased eNOS-Ser116 phosphorylation with a concomitant increase in NO production in a time-dependent manner. The amino acid sequence around the eNOS-Ser116 residue was identified as the substrate site of the regulatory subunit B56δ of protein phosphatase 2A (PP2A). As expected, okadaic acid, a specific PP2A inhibitor, reversed aphidicolin-induced eNOS-Ser116 dephosphorylation in a dose-dependent manner. Aphidicolin also increased B56δ-Ser566 phosphorylation, although expression of neither the catalytic subunit Cα (PP2A Cα) nor B56δ was altered. Ectopic expression of dominant negative (dn)-B56δ reversed all of the observed effects of aphidicolin with respect to phosphorylation of eNOS-Ser116 and B56δ-Ser566. Lastly, aphidicolin-stimulated NO production was also partially attenuated by ectopic expression of dn-B56δ. Taken together, our results are the first to demonstrate that aphidicolin decreases phosphorylation of eNOS-Ser116, at least in part by activating PP2A B56δ, resulting in NO release in BAEC.