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Hyperactivated AKT/mTOR signaling is a hallmark of pancreatic neuroendocrine tumors (PNETs). Drugs targeting this pathway are used clinically, but tumor resistance invariably develops. A better understanding of factors regulating AKT/mTOR signaling and PNET pathogenesis is needed to improve current therapies. We discovered that RABL6A, a new oncogenic driver of PNET proliferation, is required for AKT activity. Silencing RABL6A caused PNET cell-cycle arrest that coincided with selective loss of AKT-S473 (not T308) phosphorylation and AKT/mTOR inactivation. Restoration of AKT phosphorylation rescued the G1 phase block triggered by RABL6A silencing. Mechanistically, loss of AKT-S473 phosphorylation in RABL6A-depleted cells was the result of increased protein phosphatase 2A (PP2A) activity. Inhibition of PP2A restored phosphorylation of AKT-S473 in RABL6A-depleted cells, whereas PP2A reactivation using a specific small-molecule activator of PP2A (SMAP) abolished that phosphorylation. Moreover, SMAP treatment effectively killed PNET cells in a RABL6A-dependent manner and suppressed PNET growth in vivo. The present work identifies RABL6A as a new inhibitor of the PP2A tumor suppressor and an essential activator of AKT in PNET cells. Our findings offer what we believe is a novel strategy of PP2A reactivation for treatment of PNETs as well as other human cancers driven by RABL6A overexpression and PP2A inactivation., Introduction Neuroendocrine tumors (NETs) are malignancies that are clinically challenging to manage and treat whose incidence is steadily increasing (1, 2). They are slow growing, biologically diverse tumors that arise [...]

Intrinsically disordered regions (IDR) and short linear motifs (SLiMs) play pivotal roles in the intricate signaling networks governed by phosphatases and kinases. B56δ (encoded by PPP2R5D) is a regulatory subunit of protein phosphatase 2A (PP2A) with long IDRs that harbor a substrate-mimicking SLiM and multiple phosphorylation sites. De novo missense mutations in PPP2R5D cause intellectual disabilities (ID), macrocephaly, Parkinsonism, and a broad range of neurological symptoms. Our single-particle cryo-EM structures of the PP2A-B56δ holoenzyme reveal that the long, disordered arms at the B56δ termini fold against each other and the holoenzyme core. This architecture suppresses both the phosphatase active site and the substrate-binding protein groove, thereby stabilizing the enzyme in a closed latent form with dual autoinhibition. The resulting interface spans over 190 Å and harbors unfavorable contacts, activation phosphorylation sites, and nearly all residues with ID-associated mutations. Our studies suggest that this dynamic interface is coupled to an allosteric network responsive to phosphorylation and altered globally by mutations. Furthermore, we found that ID mutations increase the holoenzyme activity and perturb the phosphorylation rates, and the severe variants significantly increase the mitotic duration and error rates compared to the normal variant. [ABSTRACT FROM AUTHOR]

Vitamin A and its metabolites are known to be involved in patterning the vertebrate embryo. Study of the effect of vitamin A on axial skeletal patterning has been hindered by the fact that deficient embryos do not survive past midgestation. In this study, pregnant vitamin A-deficient rats were maintained on a purified diet containing limiting amounts of all-trans retinoic acid (12 [micro]g atRA/g diet) and given a daily oral bolus dose of retinol starting at embryonic day 0.5, 8.25, 8.5, 8.75, 9.25, 9.5, 9.75, or 10.5. Embryos were recovered at E21.5 for analysis of the skeleton and at earlier times for analysis of select mRNAs. Normal axial skeletal development and patterning were observed in embryos from pregnant animals receiving retinol starting on or before E8.75. Delay of retinol supplementation to E9.5 or later resulted in a marked increase in both occurrence and severity of skeletal malformations, extending from the craniocervical to sacral regions. Embryos from the groups receiving retinol starting at E9.5 and E9.75 had one-vertebral anterior transformations of the cervical, thoracic, lumbar, and sacral vertebrae. Few embryos survived in the E10.5 group, but these embryos yielded the most severe and extensive anteriorization events. The skeletal alterations seen in vitamin A deficiency are associated with posterior shifts in the mesodermal expression of Hoxa-4, Hoxb-3, Hoxd-3, Hoxd-4, and Hoxa-9 mRNAs, whereas the anterior domains of Hoxb-4 and Cdx2 expression are unaltered. This work defines a critical window of development in the late gastrula-stage embryo when vitamin A is essential for normal axial skeletal patterning and shows that vitamin A deficiency causes anterior homeotic transformations extending from the cervical to lumbosacral regions. Keywords: Vitamin A-deficiency; Embryonic development; Retinoic acid; Retinoid; Reproduction; Skeletal patterning; Axial patterning; Hoxd-3; Hoxb-3; Hoxd-4; Hoxb-4; Hoxa-4; Hoxa-9; Cdx2

The phosphoenolpyruvate-dependent phosphotransferase system (PTS) and the methyl-accepting chemotaxis protein McpC are found to mediate chemotaxis toward PTS substrates in Bacillus subtilis. The response to positive stimulus is primarily mediated by McpC, while components of the PTS and McpC are key factors in the duration or magnitude of the response to negative PTS carbohydrate stimulus. A multidimensional signaling mechanism involving both the PTS and specific MCPs has also been noted in the PTS' regulation of chemotaxis in B. subtilis.

Proper brain development and function requires finely controlled mechanisms for protein turnover, and disruption of genes involved in proteostasis is a common cause of neurodevelopmental disorders. Kelch-like 15 (KLHL15) is a substrate adaptor for cullin3-containing E3 ubiquitin ligases, and KLHL15 gene mutations were recently described as a cause of severe X-linked intellectual disability. Here, we used a bioinformatics approach to identify a family of neuronal microtubule-associated proteins as KLHL15 substrates, which are themselves critical for early brain development. We biochemically validated doublecortin (DCX), also an X-linked disease protein, and doublecortin-like kinase 1 and 2 as bona fide KLHL15 interactors and mapped KLHL15 interaction regions to their tandem DCX domains. Shared with two previously identified KLHL15 substrates, a FRY tripeptide at the Cterminal edge of the second DCX domain is necessary for KLHL15-mediated ubiquitination of DCX and doublecortinlike kinase 1 and 2 and subsequent proteasomal degradation. Conversely, silencing endogenous KLHL15 markedly stabilizes these DCX domain-containing proteins and prolongs their half-life. Functionally, overexpression of KLHL15 in the presence of WT DCX reduces dendritic complexity of cultured hippocampal neurons, whereas neurons expressing FRYmutant DCX are resistant to KLHL15. Collectively, our findings highlight the critical importance of the E3 ubiquitin ligase adaptor KLHL15 in proteostasis of neuronal microtubuleassociated proteins and identify a regulatory network important for development of the mammalian nervous system. [ABSTRACT FROM AUTHOR]

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. UsingB'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. [ABSTRACT FROM AUTHOR]

Mitochondrial fission catalyzed by dynamin-related protein 1 (Drp1) is necessary for mitochondrial biogenesis and maintenance of healthy mitochondria. However, excessive fission has been associated with multiple neurodegenerative disorders, and we recently reported that mice with smaller mitochondria are sensitized to ischemic stroke injury. Although pharmacological Drp1 inhibition has been put forward as neuroprotective, the specificity and mechanism of the inhibitor used is controversial. Here, we provide genetic evidence that Drp1 inhibition is neuroprotective. Drp1 is activated by dephosphorylation of an inhibitory phosphorylation site, Ser637. We identify Bβ2, a mitochondria-localized protein phosphatase 2A (PP2A) regulatory subunit, as a neuron-specific Drp1 activator in vivo Bβ2 KO mice of both sexes display elongated mitochondria in neurons and are protected from cerebral ischemic injury. Functionally, deletion of Bβ2 and maintained Drp1 Ser637 phosphorylation improved mitochondrial respiratory capacity, Ca2+ homeostasis, and attenuated superoxide production in response to ischemia and excitotoxicity in vitro and ex vivo Last, deletion of Bβ2 rescued excessive stroke damage associated with dephosphorylation of Drp1 S637 and mitochondrial fission. These results indicate that the state of mitochondrial connectivity and PP2A/Bβ2-mediated dephosphorylation of Drp1 play a critical role in determining the severity of cerebral ischemic injury. Therefore, Bβ2 may represent a target for prophylactic neuroprotective therapy in populations at high risk of stroke. [ABSTRACT FROM AUTHOR]

The self-labeling protein HaloTag has been used extensively to determine the localization and turnover of proteins of interest at the single-cell level. To this end, halogen-substituted alkanes attached to diverse fluorophores are commercially available that allow specific, irreversible labeling of HaloTag fusion proteins; however, measurement of protein of interest half-life by pulse-chase of HaloTag ligands is not widely employed because residual unbound ligand continues to label newly synthesized HaloTag fusions even after extensive washing. Excess unlabeled HaloTag ligand can be used as a blocker of undesired labeling, but this is not economical. In this study, we screened several inexpensive, low-molecular-weight haloalkanes as blocking agents in pulse-chase labeling experiments with the cell-permeable tetramethylrhodamine HaloTag ligand. We identified 7-bromoheptanol as a high-affinity, low-toxicity HaloTag-blocking agent that permits protein turnover measurements at both the cell population (by blotting) and single-cell (by imaging) levels. We show that the HaloTag pulse-chase approach is a nontoxic alternative to inhibition of protein synthesis with cycloheximide and extend protein turnover assays to long-lived proteins. [ABSTRACT FROM AUTHOR]

Highlights a study which described a theory used for the magnetization of the ferromagnetic sphere; Description of the magnetization of the ferromagnetic cube; Reference to the nucleation theory; Results of the study.

Mitochondrial fission and fusion impact numerous cellular functions and neurons are particularly sensitive to perturbations in mitochondrial dynamics. Here we describe that male mice lacking the mitochondrial A-kinase anchoring protein 1 (AKAP1) exhibit increased sensitivity in the transient middle cerebral artery occlusion model of focal ischemia. At the ultrastructural level, AKAP1-/- mice have smaller mitochondria and increased contacts between mitochondria and the endoplasmic reticulum in the brain. Mechanistically, deletion of AKAP1 dysregulates complex 11 of the electron transport chain, increases superoxide production, and impairs Ca2+ homeostasis in neurons subjected to excitotoxic glutamate. Ca2+ deregulation in neurons lacking AKAP1 can be attributed to loss of inhibitory phosphorylation of the mitochondrial fission enzyme dynamin-related protein 1 (Drpl) at the protein kinase A (PKA) site Ser637. Our results indicate that inhibition of Drpl-dependent mitochondrial fission by the outer mitochondrial AKAP 1/PKA complex protects neurons from ischemic stroke by maintaining respiratory chain activity, inhibiting superoxide production, and delaying Ca2+ deregulation. They also provide the first genetic evidence that Drp1 inhibition may be of therapeutic relevance for the treatment of stroke and neurodegeneration. [ABSTRACT FROM AUTHOR]

It seems quite justified to claim that there was significant variation in the rates virtual geomagnetic poles (VGPs) moved during the two most recent reversals of the polarity of the Earth's magnetic field. New research on this subject has shown that it is possible to gain important information on the transitional magnetic field from some sediments. Data from sediments and lava flows can provide new insights into reversal transitions. The researchers obtained and analyzed high-resolution records from sedimentary cores in the North Atlantic.

Most of the geomagnetists have dismissed the claim that the rapid changes in the Earth's main magnetic field that were recorded in two 16-million-year old lava flows at Steens Mountain in Oregon were in orders of magnitude faster than seen in direct measurements of the field. However, two of the original scientists continued their work and have shown changes up to six degrees per day. The principle of least astonishment can only be applied once one interpretation is accepted. One potential explanation is that the lava absorbed long term changes but the changes diffused slowly and so surfaced at once.

While the dynamin GTPase Drp1 plays a critical role during mitochondrial fission, mechanisms controlling its recruitment to fission sites are unclear. A current assumption is that cytosolic Drp1 is recruited directly to fission sites immediately prior to fission. Using live-cell microscopy, we find evidence for a different model, progressive maturation of Drp1 oligomers on mitochondria through incorporation of smaller mitochondrially-bound Drp1 units. Maturation of a stable Drp1 oligomer does not forcibly lead to fission. Inhibiting actin polymerization, myosin IIA, or the formin INF2 reduces both un-stimulated and ionomycin-induced Drp1 accumulation and mitochondrial fission. Actin filaments bind purified Drp1 and increase GTPase activity in a manner that is synergistic with the mitochondrial protein Mff, suggesting a role for direct Drp1/actin interaction. We propose that Drp1 is in dynamic equilibrium on mitochondria in a fission-independent manner, and that fission factors such as actin filaments target productive oligomerization to fission sites. [ABSTRACT FROM AUTHOR]

Abstract: Mitochondria are best known for their role as cellular power plants, but they also serve as signaling hubs, regulating cellular proliferation, differentiation, and survival. A kinase anchoring protein 1 (AKAP1) is a scaffold protein that recruits protein kinase A (PKA) and other signaling proteins, as well as RNA, to the outer mitochondrial membrane. AKAP1 thereby integrates several second messenger cascades to modulate mitochondrial function and associated physiological and pathophysiological outcomes. Here, we review what is currently known about AKAP1's macromolecular interactions in health and disease states, including obesity. We also discuss dynamin-related protein 1 (Drp1), the enzyme that catalyzes mitochondrial fission, as one of the key substrates of the PKA/AKAP1 signaling complex in neurons. Recent evidence suggests that AKAP1 has critical roles in neuronal development and survival, which are mediated by inhibitory phosphorylation of Drp1 and maintenance of mitochondrial integrity. [Copyright &y& Elsevier]

Fission and fusion events dynamically control the shape and function of mitochondria. The activity of the mitochondrial fission enzyme dynamin-related protein 1 (Drp1) is finely tuned by several post-translational modifications. Phosphorylation of Ser-656 by cAMP-dependent protein kinase (PKA) inhibits Drp1, whereas dephosphorylation by a mitochondrial protein phosphatase 2A isoform and the calcium-calmodulin-dependent phosphatase calcineurin (CaN) activates Drp1. Here, we identify a conserved CaN docking site on Drp1, an LXVP motif, which mediates the interaction between the phosphatase and mechanoenzyme. We mutated the LXVP motif in Drp1 to either increase or decrease similarity to the prototypical LXVP motif in the transcription factor NFAT, and assessed stability of the mutant Drp1-CaN complexes by affinity precipitation and isothermal titration calorimetry. Furthermore, we quantified effects of LXVP mutations on Drp1 dephosphorylation kinetics in vitro and in intact cells. With tools for bidirectional control of the CaN-Drp1 signaling axis in hand, we demonstrate that the Drp1 LXVP motif shapes mitochondria in neuronal and non-neuronal cells, and that CaN-mediated Drp1 dephosphorylation promotes neuronal death following oxygen-glucose deprivation. These results point to the CaN-Drp1 complex as a potential target for neuroprotective therapy of ischemic stroke. [ABSTRACT FROM AUTHOR]

Mitochondrial shape is determined by fission and fusion reactions catalyzed by large GTPases of the dynamin family, mutation of which can cause neurological dysfunction. While fission-inducing protein phosphatases have been identified, the identity of opposing kinase signaling complexes has remained elusive. We report here that in both neurons and nonneuronal cells, cAMP elevation and expression of an outer-mitochondrial membrane (OMM) targeted form of the protein kinase A (PKA) catalytic subunit reshapes mitochondria into an interconnected network. Conversely, OMM-targeting of the PKA inhibitor PKI promotes mitochondrial fragmentation upstream of neuronal death. RNAi and overexpression approaches identify mitochondria-localized A kinase anchoring protein 1 (AKAP1) as a neuroprotective and mitochondria-stabilizing factor in vitro and in vivo. According to epistasis studies with phosphorylation site-mutant dynamin-related protein 1 (Drp1), inhibition of the mitochondrial fission enzyme through a conserved PKA site is the principal mechanism by which cAMP and PKA/AKAP1 promote both mitochondrial elongation and neuronal survival. Phenocopied by a mutation that slows GTP hydrolysis, Drp1 phosphorylation inhibits the disassembly step of its catalytic cycle, accumulating large, slowly recycling Drp1 oligomers at the OMM. Unopposed fusion then promotes formation of a mitochondrial reticulum, which protects neurons from diverse insults. [ABSTRACT FROM AUTHOR]

Protein phosphatase 2A- (PP2A-) catalyzed dephosphorylation of target substrate proteins is widespread and critical for cellular function. PP2A is predominantly found as a heterotrimeric complex of a catalytic subunit (C), a scaffolding subunit (A), and one member of 4 families of regulatory subunits (B). Substrate specificity of the holoenzyme complex is determined by the subcellular locale the complex is confined to, selective incorporation of the B subunit, interactions with endogenous inhibitory proteins, and specific intermolecular interactions between PP2A and target substrates. Here, we discuss recent studies that have advanced our understanding of the molecular determinants for PP2A substrate specificity. [ABSTRACT FROM AUTHOR]

The time-average geomagnetic field is commonly assumed to be a geocentric axial dipole field (GAD). This assumption is crucial to many subfields of paleomagnetism in which one needs to determine paleolatitude. In some cases an interval of only 104 years is sufficient to obtain the time-average field, while in others an interval of at least 105 years is required. Numerous spherical harmonic models for the time-average field for the past 5 Myr exhibit conflicting estimates for non-dipole terms. New criteria, most notably that the Gauss coefficients should exhibit the same time-average magnitudes for reverse and normal polarity states, are used to evaluate these models. We find that the GAD assumption is surprisingly good because of the character of temporal variations in the non-dipole field. In the time-average field, other than the GAD field, only a small axial quadrupole field seems to survive careful scrutiny. If the existence of such a time-average quadrupole field is confirmed, it probably reflects coupling of some form between the dipole and quadrupole families. We find no convincing evidence in the Gauss coefficients for longitudinal variation of the time-average field, but there is some evidence for such asymmetry in the paleosecular variation data. [Copyright &y& Elsevier]

Virtual dipole moments (VDM) from the past 320 million years show bimodal distributions when averaged over time intervals of several tens of millions of years. The two peaks in the bimodal distributions occur at about (4–5)×1022 A m2 and at about twice that value, these two values vary little with time. However, the relative contributions of the two modes do vary significantly with time. It is possible that one of these modes is associated with an as yet unidentified rock magnetic artifact. Alternatively, the observation admits the intriguing interpretation that as well as having two polarity states the geodynamo has two (relatively constant) intensity states. Our perception of the paleofield intensity, and its variation, in a given time interval then depends on how much time the dynamo has spent in each of the intensity states and on how well our observations have sampled this. [Copyright &y& Elsevier]

Neurodevelopmental disorders (NDDs), including intellectual disability (ID), autism and schizophrenia, have high socioeconomic impact, yet poorly understood etiologies. A recent surge of large-scale genome or exome sequencing studies has identified a multitude of mostly de novo mutations in subunits of the protein phosphatase 2A (PP2A) holoenzyme that are strongly associated with NDDs. PP2A is responsible for at least 50% of total Ser/Thr dephosphorylation in most cell types and is predominantly found as trimeric holoenzymes composed of catalytic (C), scaffolding (A) and variable regulatory (B) subunits. PP2A can exist in nearly 100 different subunit combinations in mammalian cells, dictating distinct localizations, substrates and regulatory mechanisms. PP2A is well established as a regulator of cell division, growth, and differentiation, and the roles of PP2A in cancer and various neurodegenerative disorders, such as Alzheimer's disease, have been reviewed in detail. This Review summarizes and discusses recent reports on NDDs associated with mutations of PP2A subunits and PP2A-associated proteins. We also discuss the potential impact of these mutations on the structure and function of the PP2A holoenzymes and the etiology of NDDs. [ABSTRACT FROM AUTHOR]