PP2A Phosphatase as an Emerging Viral Host Factor.
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ABSTRACT:
SUBMITTER: Barski MS
PROVIDER: S-EPMC8371333 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The widespread reorganization of cellular architecture in mitosis is achieved through extensive protein phosphorylation, driven by the coordinated activation of a mitotic kinase network and repression of counteracting phosphatases. Phosphatase activity must subsequently be restored to promote mitotic exit. Although Cdc14 phosphatase drives this reversal in budding yeast, protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) activities have each been independently linked to mitotic exit control in other eukaryotes. Here we describe a mitotic phosphatase relay in which PP1 reactivation is required for the reactivation of both PP2A-B55 and PP2A-B56 to coordinate mitotic progression and exit in fission yeast. The staged recruitment of PP1 (the Dis2 isoform) to the regulatory subunits of the PP2A-B55 and PP2A-B56 (B55 also known as Pab1; B56 also known as Par1) holoenzymes sequentially activates each phosphatase. The pathway is blocked in early mitosis because the Cdk1-cyclin B kinase (Cdk1 also known as Cdc2) inhibits PP1 activity, but declining cyclin B levels later in mitosis permit PP1 to auto-reactivate. PP1 first reactivates PP2A-B55; this enables PP2A-B55 in turn to promote the reactivation of PP2A-B56 by dephosphorylating a PP1-docking site in PP2A-B56, thereby promoting the recruitment of PP1. PP1 recruitment to human, mitotic PP2A-B56 holoenzymes and the sequences of these conserved PP1-docking motifs suggest that PP1 regulates PP2A-B55 and PP2A-B56 activities in a variety of signalling contexts throughout eukaryotes.
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Project description:Autosomal dominant polycystic kidney disease (ADPKD) is associated with a number of cellular defects such as hyperproliferation, apoptosis, and dedifferentiation. Mutations in polycystin-1 (PC1) account for ?85% of ADPKD. Here, we showed that wild-type (WT) or mutant PC1 composed of the last five transmembrane (TM) domains and the C-terminus (termed PC1-5TMC) inhibits cell proliferation and protein translation, as well as the downstream effectors of mTOR, consistent with previous reports. Knockdown of B56?, a subunit of the protein phosphatase 2A (PP2A) complex, or application of PP2A inhibitor okadaic acid or calyculin A, abolished the inhibitory effect of PC1 and PC1-5TMC on proliferation, indicating that PP2A/B56? mediates the regulation of cell proliferation by PC1. In addition to the phosphorylated S6 and 4EBP1, B56? was also downregulated by PC1 and PC1-5TMC. Furthermore, the downregulation of B56?, which may be mediated by mTOR but not AKT, can account for the dependence of PC1-inhibited proliferation on PP2A.
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Project description:The activity and specificity of serine/threonine phosphatases are governed largely by their associated proteins. alpha4 is an evolutionarily conserved noncatalytic subunit for PP2A-like phosphatases. Though alpha4 binds to only a minority of PP2A-related catalytic subunits, alpha4 deletion leads to progressive loss of all PP2A, PP4, and PP6 phosphatase complexes. In healthy cells, association with alpha4 renders catalytic (C) subunits enzymatically inactive while protecting them from proteasomal degradation until they are assembled into a functional phosphatase complex. During cellular stress, existing PP2A complexes can become unstable. Under such conditions, alpha4 sequesters released C subunits and is required for the adaptive increase in targeted PP2A activity that can dephosphorylate stress-induced phosphorylated substrates. Consistent with this, overexpression of alpha4 protects cells from a variety of stress stimuli, including DNA damage and nutrient limitation. These findings demonstrate that alpha4 plays a required role in regulating the assembly and maintenance of adaptive PP2A phosphatase complexes.
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Project description:Protein phosphatase 2A (PP2A) is one of the most abundant serine-threonine phosphatases in mammalian cells. PP2A is a hetero-trimeric holoenzyme participating in a variety of physiological processes whose deregulation is often associated to cancer. The specificity and activity of this phosphatase is tightly modulated by a family of regulatory B subunits that dock the catalytic subunit to the substrates. Here we characterize a novel and unconventional molecular mechanism controlling the activity of the tumor suppressor PP2A. By applying a mass spectrometry-based interactomics approach, we identified novel PP2A interacting proteins. Unexpectedly we found that a significant number of RAB proteins associate with the PP2A scaffold subunit (PPP2R1A), but not with the catalytic subunit (PPP2CA). Such interactions occur in vitro and in vivo in specific subcellular compartments. Notably we demonstrated that one of these RAB proteins, RAB9, competes with the catalytic subunit PPP2CA in binding to PPP2R1A. This competitive association has an important role in controlling the PP2A catalytic activity, which is compromised in several solid tumors and leukemias.
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Project description:EG5 (KIF11) is a member of the kinesin-like protein family involved in centrosome separation and bipolar spindle formation. When a cell enters mitosis, CDK1 phosphorylates EG5 at Thr926 and promotes EG5 localization on the mitotic spindle which drives bipolar spindle formation. EG5 provides power for spindle movement and thus controls the dynamics of spindle assembly. However, little is known about EG5 regulation or how EG5 detaches from the spindle upon mitotic exit. In this study we identify EG5 as a novel substrate of PP2A phosphatase, and we show that the PP2A/B55α complex plays an important role in mitotic exit by a mechanism involving EG5. The PP2A/B55α complex physically associates with the EG5 C-terminal tail domain and dephosphorylates EG5 at Thr926 that enables mitotic exit. Conversely PP2A knockdown cells show a high level of phospho-EG5 in late metaphase, which is associated with a delay in mitotic exit. These phenotypic features are similar to those induced by EG5/T926D transfection that mimics phosphorylated EG5 status. Our results argue that PP2A controls mitotic exit through EG5 dephosphorylation. Lack of PP2A leads to abnormal EG5 activation, resulting in delay of mitotic exit.
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Project description:Protein phosphatase 2A (PP2A) is a large enzyme family responsible for most cellular Ser/Thr dephosphorylation events. PP2A substrate specificity, localization, and regulation by second messengers rely on more than a dozen regulatory subunits (including B/R2, B'/R5, and B″/R3), which form the PP2A heterotrimeric holoenzyme by associating with a dimer comprising scaffolding (A) and catalytic (C) subunits. Because of partial redundancy and high endogenous expression of PP2A holoenzymes, traditional approaches of overexpressing, knocking down, or knocking out PP2A regulatory subunits have yielded only limited insights into their biological roles and substrates. To this end, here we sought to reduce the complexity of cellular PP2A holoenzymes. We used tetracycline-inducible expression of pairs of scaffolding and regulatory subunits with complementary charge-reversal substitutions in their interaction interfaces. For each of the three regulatory subunit families, we engineered A/B charge-swap variants that could bind to one another, but not to endogenous A and B subunits. Because endogenous Aα was targeted by a co-induced shRNA, endogenous B subunits were rapidly degraded, resulting in expression of predominantly a single PP2A heterotrimer composed of the A/B charge-swap pair and the endogenous catalytic subunit. Using B'δ/PPP2R5D, we show that PP2A complexity reduction, but not PP2A overexpression, reveals a role of this holoenzyme in suppression of extracellular signal-regulated kinase signaling and protein kinase A substrate dephosphorylation. When combined with global phosphoproteomics, the PP2A/B'δ reduction approach identified consensus dephosphorylation motifs in its substrates and suggested that residues surrounding the phosphorylation site play roles in PP2A substrate specificity.
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Project description:Phosphatase PP2A expression levels are positively correlated to the clinical severity of systemic lupus erythematosus (SLE) and IL17A cytokine overproduction, indicating a potential role of PP2A in controlling TH17 differentiation and inflammation. By generating a mouse strain with ablation of the catalytic subunit α of PP2A in peripheral mature T cells (PP2A cKO), we demonstrate that the PP2A complex is essential for TH17 differentiation. These PP2A cKO mice had reduced TH17 cell numbers and less severe disease in an experimental autoimmune encephalomyelitis (EAE) model. PP2A deficiency also ablated C-terminal phosphorylation of SMAD2 but increased C-terminal phosphorylation of SMAD3. By regulating the activity of RORγt via binding, the changes in the phosphorylation status of these R-SMADs reduced Il17a gene transcription. Finally, PP2A inhibitors showed similar effects on TH17 cells as were observed in PP2A cKO mice, i.e., decreased TH17 differentiation and relative protection of mice from EAE. Taken together, these data demonstrate that phosphatase PP2A is essential for TH17 differentiation and that inhibition of PP2A could be a possible therapeutic approach to controlling TH17-driven autoimmune diseases.
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