Phosphorylation Dynamics in a flg22-Induced, G Protein-Dependent Network Reveals the AtRGS1 Phosphatase.

The microbe-associated molecular pattern flg22 is recognized in a flagellin-sensitive 2-dependent manner in root tip cells. Here, we show a rapid and massive change in protein abundance and phosphorylation state of the Arabidopsis root cell proteome in WT and a mutant deficient in heterotrimeric G-protein-coupled signaling. flg22-induced changes fall on proteins comprising a subset of this proteome, the heterotrimeric G protein interactome, and on highly-populated hubs of the immunity network. Approximately 95% of the phosphorylation changes in the heterotrimeric G-protein interactome depend, at least partially, on a functional G protein complex. One member of this interactome is ATBα, a substrate-recognition subunit of a protein phosphatase 2A complex and an interactor to Arabidopsis thaliana Regulator of G Signaling 1 protein (AtRGS1), a flg22-phosphorylated, 7-transmembrane spanning modulator of the nucleotide-binding state of the core G-protein complex. A null mutation of ATBα strongly increases basal endocytosis of AtRGS1. AtRGS1 steady-state protein level is lower in the atbα mutant in a proteasome-dependent manner. We propose that phosphorylation-dependent endocytosis of AtRGS1 is part of the mechanism to degrade AtRGS1, thus sustaining activation of the heterotrimeric G protein complex required for the regulation of system dynamics in innate immunity. The PP2A(ATBα) complex is a critical regulator of this signaling pathway.

PP2ACdc55 Phosphatase Imposes Ordered Cell-Cycle Phosphorylation by Opposing Threonine Phosphorylation.

In the quantitative model of cell-cycle control, progression from G1 through S phase and into mitosis is ordered by thresholds of increasing cyclin-dependent kinase (Cdk) activity. How such thresholds are read out by substrates that respond with the correct phosphorylation timing is not known. Here, using the budding yeast model, we show that the abundant PP2ACdc55 phosphatase counteracts Cdk phosphorylation during interphase and delays phosphorylation of late Cdk substrates. PP2ACdc55 specifically counteracts phosphorylation on threonine residues, and consequently, we find that threonine-directed phosphorylation occurs late in the cell cycle. Furthermore, the late phosphorylation of a model substrate, Ndd1, depends on threonine identity of its Cdk target sites. Our results support a model in which Cdk-counteracting phosphatases contribute to cell-cycle ordering by imposing Cdk thresholds. They also unveil a regulatory principle based on the phosphoacceptor amino acid, which is likely to apply to signaling pathways beyond cell-cycle control.

Repression of the Aryl Hydrocarbon Receptor Is Required to Maintain Mitotic Progression and Prevent Loss of Pluripotency of Embryonic Stem Cells.

Lack of cell cycle checkpoints and uninterrupted passage through S-phase continuously renew the embryonic stem (ES) cell population and maintain pluripotency. Here, we show that to regulate mitotic progression and pluripotency ES cells must keep the aryl hydrocarbon receptor (AHR), an environmental sensor and transcriptional regulator, in a persistent state of repression. This repression, however, is not always absolute, causing the AHR to fluctuate between reversible states of expression and repression, with a fraction of the cells escaping repression at any one time. Cells that escape AHR repression exhibit reduced levels of the pluripotency factors OCT4 and SOX2 and show an extended mitotic traverse time due to AHR-dependent MID1 repression and the subsequent disruption of the MID1-PP2A-CDC25B-CDK1 signaling pathway that regulates mitosis. Unlike the bulk of the cell population that differentiates into cardiomyocytes upon stimulation, AHR-expressing ES cells restrict cardiogenesis and commit to a neuroglia cell fate. It appears that the untimely expression of the Ahr gene needs to be repressed to maintain ES cell mitotic progression and prevent premature loss of pluripotency. Stem Cells 2016;34:2825-2839.

Protein phosphatase PP2A - a novel interacting partner of carnitine transporter OCTN2 (SLC22A5) in rat astrocytes.

l-Carnitine is essential for translocation of fatty acids for their mitochondrial ß-oxidation, a process shown in the brain to take place in astrocytes. Organic cation and carnitine plasma membrane transporter OCTN2 (SLC22A5) is present in astrocytes. OCTN2 activity and localization were previously shown to be regulated by protein kinase C (PKC), although no phosphorylation of the transporter was detected. In this study, mass spectrometry was used to identify rOctn2-interacting partners in astrocytes: several cytoskeletal, ribosomal, mitochondrial, heat-shock proteins, as well as proteins involved in trafficking and signaling pathways. The analysis of signaling proteins shows that Octn2 co-precipitated with PP2A phosphatase catalytical (C) and structural (A) subunits, and with its regulatory B"' subunits - striatin, SG2NA, and zinedin. The Octn2/PP2A complex is mainly detected in endoplasmic reticulum. PKC activation increases both, carnitine transport and, as shown by immunofluorescence and surface biotinylation, transporter presence in plasma membrane. It also results in phosphorylation of SG2NA, zinedin, and catalytical subunit, although co-precipitation, immunocytochemistry, and proximity ligation assay experiments showed that only the amount of SG2NA decreased in the complex with Octn2. PP2A inhibition with okadaic acid does not lead to Octn2 phosphorylation; however, it abolishes observed effects of PKC activation. We postulate that PKC phosphorylates SG2NA, resulting in its dissociation from the complex and transfer of Octn2 to the plasma membrane, leading to increased transporter activity. The observed interaction could affect brain functioning in vivo, both in fatty acid metabolism and in control of carnitine homeostasis, known to change in certain brain pathologies.

Preparing a cell for nuclear envelope breakdown: Spatio-temporal control of phosphorylation during mitotic entry.

Chromosome segregation requires the ordered separation of the newly replicated chromosomes between the two daughter cells. In most cells, this requires nuclear envelope (NE) disassembly during mitotic entry and its reformation at mitotic exit. Nuclear envelope breakdown (NEB) results in the mixture of two cellular compartments. This process is controlled through phosphorylation of multiple targets by cyclin-dependent kinase 1 (Cdk1)-cyclin B complexes as well as other mitotic enzymes. Experimental evidence also suggests that nucleo-cytoplasmic transport of critical cell cycle regulators such as Cdk1-cyclin B complexes or Greatwall, a kinase responsible for the inactivation of PP2A phosphatases, plays a major role in maintaining the boost of mitotic phosphorylation thus preventing the potential mitotic collapse derived from NEB. These data suggest the relevance of nucleo-cytoplasmic transport not only to communicate cytoplasmic and nuclear compartments during interphase, but also to prepare cells for the mixture of these two compartments during mitosis.

Structural, enzymatic and spatiotemporal regulation of PP2A-B55 phosphatase in the control of mitosis.

Cells require major physical changes to induce a proper repartition of the DNA. Nuclear envelope breakdown, DNA condensation and spindle formation are promoted at mitotic entry by massive protein phosphorylation and reversed at mitotic exit by the timely and ordered dephosphorylation of mitotic substrates. This phosphorylation results from the balance between the activity of kinases and phosphatases. The role of kinases in the control of mitosis has been largely studied, however, the impact of phosphatases has long been underestimated. Recent data have now established that the regulation of phosphatases is crucial to confer timely and ordered cellular events required for cell division. One major phosphatase involved in this process is the phosphatase holoenzyme PP2A-B55. This review will be focused in the latest structural, biochemical and enzymatic insights provided for PP2A-B55 phosphatase as well as its regulators and mechanisms of action.

PP2A subunit PPP2R2C is downregulated in the brains of Alzheimer's transgenic mice.

Targeting of PP2A suggests a close link to tau-related cognitive and functional declines. However, little is known about how the expression of PP2A subunits and PP2A activity are dysregulated in the course of AD, precluding any specific targeting strategy for restoring PP2A in AD patients. Although the PP2A heterotrimer containing the regulatory subunit PR55/Bα (encoded by the PPP2R2A gene) is the major tau phosphatase, the involvement of other brain-specific PP2A regulatory subunits in tau dephosphorylation remains unknown. PR55/Bγ (encoded by the PPP2R2C gene) is a pivotal phosphatase in the brain, and single-nucleotide polymorphisms (SNPs) of PPP2R2C are involved in several mental disorders. By measuring the differential spatiotemporal expression patterns of PPP2R2C in Wt and transgenic AD mice, we revealed that PPP2R2C expression is downregulated in the aged AD mouse brain as compared to the Wt mouse brain. In cultured cells, PPP2R2C expression regulates PP2A activity and tau dephosphorylation. These results suggest that dysregulation of PPP2R2C expression may be involved in the onset of AD and that specifically targeting PPP2R2C expression or activity is a promising strategy against brain dementia disorders, including AD and other tauopathies.

Kinetochore Recruitment of the Spindle and Kinetochore-Associated (Ska) Complex Is Regulated by Centrosomal PP2A in Caenorhabditis elegans.

During mitosis, kinetochore-microtubule interactions ensure that chromosomes are accurately segregated to daughter cells. RSA-1 (regulator of spindle assembly-1) is a regulatory B″ subunit of protein phosphatase 2A that was previously proposed to modulate microtubule dynamics during spindle assembly. We have identified a genetic interaction between the centrosomal protein, RSA-1, and the spindle- and kinetochore-associated (Ska) complex in Caenorhabditis elegans In a forward genetic screen for suppressors of rsa-1(or598) embryonic lethality, we identified mutations in ska-1 and ska-3 Loss of SKA-1 and SKA-3, as well as components of the KMN (KNL-1/MIS-12/NDC-80) complex and the microtubule end-binding protein EBP-2, all suppressed the embryonic lethality of rsa-1(or598) These suppressors also disrupted the intracellular localization of the Ska complex, revealing a network of proteins that influence Ska function during mitosis. In rsa-1(or598) embryos, SKA-1 is excessively and prematurely recruited to kinetochores during spindle assembly, but SKA-1 levels return to normal just prior to anaphase onset. Loss of the TPX2 homolog, TPXL-1, also resulted in overrecruitment of SKA-1 to the kinetochores and this correlated with the loss of Aurora A kinase on the spindle microtubules. We propose that rsa-1 regulates the kinetochore localization of the Ska complex, with spindle-associated Aurora A acting as a potential mediator. These data reveal a novel mechanism of protein phosphatase 2A function during mitosis involving a centrosome-based regulatory mechanism for Ska complex recruitment to the kinetochore.

Patterning and gastrulation defects caused by the tw18 lethal are due to loss of Ppp2r1a.

The mouse t haplotype, a variant 20 cM genomic region on Chromosome 17, harbors 16 embryonic control genes identified by recessive lethal mutations isolated from wild mouse populations. Due to technical constraints so far only one of these, the tw5 lethal, has been cloned and molecularly characterized. Here we report the molecular isolation of the tw18 lethal. Embryos carrying the tw18 lethal die from major gastrulation defects commencing with primitive streak formation at E6.5. We have used transcriptome and marker gene analyses to describe the molecular etiology of the tw18 phenotype. We show that both WNT and Nodal signal transduction are impaired in the mutant epiblast, causing embryonic patterning defects and failure of primitive streak and mesoderm formation. By using a candidate gene approach, gene knockout by homologous recombination and genetic rescue, we have identified the gene causing the tw18 phenotype as Ppp2r1a, encoding the PP2A scaffolding subunit PR65alpha. Our work highlights the importance of phosphatase 2A in embryonic patterning, primitive streak formation, gastrulation, and mesoderm formation downstream of WNT and Nodal signaling.

Homeostatic control of START through negative feedback between Cln3-Cdk1 and Rim15/Greatwall kinase in budding yeast.

How cells coordinate growth and division is key for size homeostasis. Phosphorylation by G1-CDK of Whi5/Rb inhibitors of SBF/E2F transcription factors triggers irreversible S-phase entry in yeast and metazoans, but why this occurs at a given cell size is not fully understood. We show that the yeast Rim15-Igo1,2 pathway, orthologous to Gwl-Arpp19/ENSA, is up-regulated in early G1 and helps promoting START by preventing PP2ACdc55 to dephosphorylate Whi5. RIM15 overexpression lowers cell size while IGO1,2 deletion delays START in cells with low CDK activity. Deletion of WHI5, CDC55 and ectopic CLN2 expression suppress the START delay of igo1,2∆ cells. Rim15 activity increases after cells switch from fermentation to respiration, where Igo1,2 contribute to chromosome maintenance. Interestingly Cln3-Cdk1 also inhibits Rim15 activity, which enables homeostatic control of Whi5 phosphorylation and cell cycle entry. We propose that Rim15/Gwl regulation of PP2A plays a hitherto unappreciated role in cell size homeostasis during metabolic rewiring of the cell cycle.