Endogenous Protein Phosphatase Research Articles

Ellman's reagent prevents dephosphorylation of histones during isolation of mitotic chromosomes.

Histones H1 and H3 are highly phosphorylated in mitotic HeLa cells but are rapidly dephosphorylated by endogenous protein phosphatases during the isolation of metaphase chromosomes. We show that this dephosphorylation can be prevented by including the sulfhydryl reagent 5,5'-dithiobis-(2-nitrobenzoate) (Ellman's reagent, or DTNB) in the isolation buffer. The minimal amount of DTNB required is approximately stoichiometric with the number of sulfhydryl groups in the lysate. Inhibition of the protein phosphatases can subsequently be reversed by treatment with dithiothreitol or 2-mercaptoethanol. DTNB is compatible with the isolation of either metaphase chromosome clusters or individual metaphase chromosomes. It should be useful in investigations of the structure and biochemistry ofchromatin and chromosomes and in the study of possible functions for mitotic histone phosphorylation.

Activation of endogenous protein phosphatase 1 enhances the calcium sensitivity of the ryanodine receptor type 2 in murine ventricular cardiomyocytes.

Increased protein phosphatase 1 (PP-1) activity has been found in end stage human heart failure. Although PP-1 has been extensively studied, a detailed understanding of its role in the excitation-contraction coupling mechanism, in normal and diseased hearts, remains elusive. The present study investigates the functional effect of the PP-1 activity on local Ca2+ release events in ventricular cardiomyocytes, by using an activating peptide (PDP3) for the stimulation of the endogenous PP-1 protein. We report that acute de-phosphorylation may increase the sensitivity of RyR2 channels to Ca2+ in situ, and that the RyR2-serine2808 phosphorylation site may mediate such a process. Our approach unmasks the functional importance of PP-1 in the regulation of RyR2 activity, suggesting a potential role in the generation of a pathophysiological sarcoplasmic reticulum Ca2+ leak in the diseased heart. Changes in cardiac ryanodine receptor (RyR2) phosphorylation are considered to be important regulatory and disease related post-translational protein modifications. The extent of RyR2 phosphorylation is mainly determined by the balance of the activities of protein kinases and phosphatases, respectively. Increased protein phosphatase-1 (PP-1) activity has been observed in heart failure, although the regulatory role of this enzyme on intracellular Ca2+ handling remains poorly understood. To determine the physiological and pathophysiological significance of increased PP-1 activity, we investigated how the PP-1 catalytic subunit (PP-1c) alters Ca2+ sparks in permeabilized cardiomyocytes and we also applied a PP-1-disrupting peptide (PDP3) to specifically activate endogenous PP-1, including the one anchored on the RyR2 macromolecular complex. We compared wild-type and transgenic mice in which the usually highly phosphorylated site RyR2-S2808 has been ablated to investigate its involvement in RyR2 modulation (S2808A+/+ ). In wild-type myocytes, PP-1 increased Ca2+ spark frequency by two-fold, followed by depletion of the sarcoplasmic reticulum Ca2+ store. Similarly, PDP3 transiently increased spark frequency and decreased sarcoplasmic reticulum Ca2+ load. RyR2 Ca2+ sensitivity, which was assessed by Ca2+ spark recovery analysis, was increased in the presence of PDP3 compared to a negative control peptide. S2808A+/+ cardiomyocytes did not respond to both PP-1c and PDP3 treatment. Our results suggest an increased Ca2+ sensitivity of RyR2 upon de-phosphorylation by PP-1. Furthermore, we have confirmed the S2808 site as a target for PP-1 and as a potential link between RyR2s modulation and the cellular response.

Temperature and serine phosphorylation regulate glycerol-3-phosphate dehydrogenase in skeletal muscle of hibernating Richardson’s ground squirrels

Glycerol-3-phosphate dehydrogenase (G3PDH) bridges carbohydrate and lipid metabolism by interconverting glycerol-3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). This reversible reaction converts G3P derived from triglyceride hydrolysis to DHAP that can then enter glycolysis or gluconeogenesis and, in the reverse reaction, makes G3P for use in triglyceride biosynthesis. Small hibernating mammals rely almost exclusively on triglyceride reserves as their fuel for energy production during torpor and the recovery of glycerol after lipolysis is an important source of carbohydrate over the nonfeeding winter months. G3PDH (∼37 kDa) was purified from skeletal muscle of euthermic and hibernating Richardson's ground squirrels (Urocitellus richardsonii) using three column chromatography steps. Analysis of enzyme kinetic properties revealed that G3PDH from hibernator muscle had higher affinities for G3P and NAD at low (5 °C) assay temperature compared with high (21 or 37 °C) and a greater stability in the presence of denaturing agents (urea, guanidine hydrochloride) or high temperature (50 °C). Immunoblotting showed that hibernator muscle G3PDH had a higher phosphoserine content than the enzyme from euthermic controls and incubation studies showed that enzyme affinity for G3P changed significantly by stimulating endogenous protein kinases or phosphatases. Overall, this study suggests that the properties of ground squirrel muscle G3PDH are modulated by temperature and post-translational phosphorylation to alter enzyme function under euthermic versus hibernating states.

Increased transcript levels and kinetic function of pyruvate kinase during severe dehydration in aestivating African clawed frogs, Xenopus laevis.

The African clawed frog, Xenopus laevis, can withstand extremely arid conditions through aestivation, resulting in dehydration and urea accumulation. Aestivating X. laevis reduce their metabolic rate, and rely on anaerobic glycolysis to meet reduced ATP demands. The present study investigated how severe dehydration affected the transcript levels, kinetic profile, and phosphorylation state of the key glycolytic enzyme pyruvate kinase (PK) in the liver and skeletal muscle of X. laevis. Compared to control frogs, severely dehydrated frogs showed an increase in the transcript abundance of both liver and muscle isoforms of PK. While the kinetics of muscle PK did not differ between dehydrated and control frogs, PK from the liver of dehydrated frogs had a lower Km for phosphoenolpyruvate (PEP) (38%), a lower Ka for fructose-1,6-bisphosphate (F1,6P2) (32%), and a greater activation of PK via F1,6P2 (1.56-fold). PK from dehydrated frogs also had a lower phosphorylation-state (25%) in comparison to the enzyme from control frogs in the liver. Experimental manipulation of the phosphorylation-state of liver PK taken from control frogs by endogenous protein phosphatases resulted in decreased phosphorylation, and a similar kinetic profile as seen in dehydrated frogs. The physiological consequence of dehydration-induced PK modification appears to adjust PK function to remain active during a metabolically depressed state. This study provides evidence for the maintenance of PK activity through elevated mRNA levels and a dephosphorylation event which activates frog liver PK in the dehydrated state in order to facilitate the production of ATP via anaerobic glycolysis.

Overexpression of CIP2A is associated with poor prognosis in multiple myeloma

Cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous protein phosphatase 2A (PP2A) inhibitor, has been identified as an oncoprotein in promoting cancer initiation and progression of several types of cancer. However, the expression and the role played by CIP2A in the pathogenesis of multiple myeloma (MM) remain unclear. In this study, we showed that CIP2A was overexpressed in human MM cell lines and MM patients’ bone marrow tissues. Clinicopathologic analysis showed that CIP2A expression was significantly correlated with clinical stage and percent of plasma cells in bone marrow. Kaplan–Meier analysis revealed that patients with high CIP2A expression presented with poorer overall survival rates than those with low CIP2A expression. Moreover, CIP2A knockdown in MM cells resulted in attenuated proliferative abilities. In addition, CIP2A depletion sensitizes dexamethasone (Dex)-resistant cells to Dex. The effect of CIP2A on proliferation and Dex therapy was mediated by the inhibition of PP2A, which in turn activated Akt. In vivo studies confirmed that CIP2A regulated MM tumorigenesis and the phosphorylation of Akt. Taken together, our results suggest that CIP2A oncoprotein plays an important role in MM progression and could serve as a prognosis marker and a novel therapeutic target for the treatment of patients with MM.

Interrogating Endogenous Protein Phosphatase Activity with Rationally Designed Chemosensors.

We introduce a versatile approach for repurposing protein kinase chemosensors, containing the phosphorylation-sensitive sulfonamido-oxine fluorophore termed Sox, for the specific determination of endogenous protein phosphatase activity from whole cell lysates and tissue homogenates. As a demonstration of this approach, we design and evaluate a direct chemosensor for protein tyrosine phosphatase-1B (PTP1B), an established signaling node in human disease. The optimal sensor design is capable of detecting as little as 6 pM (12 pg) full-length recombinant PTP1B and is remarkably selective for PTP1B among a panel of highly homologous tyrosine phosphatases. Coupling this robust activity probe with the specificity of antibodies allowed for the temporal analysis of endogenous PTP1B activity dynamics in lysates generated from HepG2 cells after stimulation with insulin. Lastly, we leveraged this assay format to profile PTP1B activity perturbations in a rat model of nonalcoholic fatty liver disease (NAFLD), providing direct evidence for elevated PTP1B catalytic activity in this disease state. Given the modular nature of this assay, we anticipate that this approach will have broad utility in monitoring phosphatase activity dynamics in human disease states.

Abstract 3350: CIP2A regulates cell proliferation via akt signaling pathway in human lung cancer

Abstract CIP2A is an intracellular endogenous protein phosphatase 2A (PP2A) inhibitor with oncogenic activities. CIP2A was initially identified as a tumor-associated antigen (TAA) in gastric and liver cancer patients, and it was overexpressed in a variety of cancer types. Recently, we found that CIP2A was overexpressed in human lung cancer specimens as compared to normal lung tissues. The elevated expression of CIP2A in lung cancer cells results in an increased cell proliferation. The observation on the association between CIP2A and AKT phosphorylation at S473 in our lab and other labs suggested the role of CIP2A in regulating AKT phosphorylation. Our preliminary data indicated that CIP2A might promote cell proliferation through AKT signaling pathway under growth factor stimulation. Interestingly, the depletion of CIP2A in lung cancer cells did not induce a global change of AKT phosphatase activity, which implied that CIP2A might recognize specific AKT targets and play certain roles in the signaling pathway. In this study, we identified several AKT substrates specifically targeted by CIP2A, such as Hsp70 and mTOR, by proteomic-based systematic screening. The candidates obtained from our study will expand the understanding on protein-substrate interaction and therefore direct the cancer drug design. Therefore, our study addressed a novel role of CIP2A in mediating cancer progression through interacting with AKT pathway in a substrate-dependent manner. Citation Format: Ningjing Lei, Bo Peng, Jiangying Zhang. CIP2A regulates cell proliferation via akt signaling pathway in human lung cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3350. doi:10.1158/1538-7445.AM2014-3350

CIP2A regulates cell proliferation via the AKT signaling pathway in human lung cancer.

Cancerous inhibitor of PP2A (CIP2A) is an intracellular endogenous protein phosphatase 2A (PP2A) inhibitor with oncogenic activities. Initially identified as a tumor-associated antigen (TAA) in gastric and liver cancer patients, CIP2A was overexpressed in a variety of cancer types. The overexpression of CIP2A in cancer cells is associated with increased cell proliferation. However, the mechanism of CIP2A in cancer cell proliferation remains poorly understood. In the present study, we reported that CIP2A can regulate AKT phosphorylation at S473 under growth factor stimulation and our results also showed that CIP2A may promote cell proliferation through the AKT signaling pathway. Notably, depletion of CIP2A did not induce a global change of AKT phosphatase activity, which indicated that CIP2A may recognize specific AKT targets and play certain roles in the signaling pathway. In addition, we detected that CIP2A expression was associated with mTOR phosphorylation. Our further analysis corroborated the relationship between CIP2A and AKT-mTOR signaling pathway. Therefore, our study addressed a novel role of CIP2A in mediating cancer progression through interacting with the AKT-mTOR signaling pathway.

Characterization of SET/I2PP2A Isoforms in Dogs

ABSTRACTSET is an endogenous protein phosphatase 2A (PP2A) inhibitor and is associated with a poor prognosis in human leukemia. Previously, we reported increased SET protein levels in canine lymphoma cell lines and the potential therapeutic application of SET antagonists in canine lymphoma. Here, we found that canine cells express several isoforms of the SET protein. We cloned 4 isoforms of SET, named SETα, β, γ and δ. Genomic BLAST showed that the SET genes are located on chromosomes X, 7, 1 and 8, respectively. An immunofluorescent study showed nuclear localization of SETα and β, and nuclear and cytosolic localization of SETγ and δ. We confirmed that SETα and β possess the ability to associate with PP2A. Our data reveal the existence of unique SET isoforms that should be taken into account in SET-targeting drug development studies in dogs.

Protein phosphatase 4 is phosphorylated and inactivated by Cdk in response to spindle toxins and interacts with γ-tubulin

Many pharmaceuticals used to treat cancer target the cell cycle or mitotic spindle dynamics, such as the anti-tumor drug, paclitaxel, which stabilizes microtubules. Here we show that, in cells arrested in mitosis with the spindle toxins, nocodazole, or paclitaxel, the endogenous protein phosphatase 4 (Ppp4) complex Ppp4c-R2-R3A is phosphorylated on its regulatory (R) subunits, and its activity is inhibited. The phosphorylations are blocked by roscovitine, indicating that they may be mediated by Cdk1-cyclin B. Endogenous Ppp4c is enriched at the centrosomes in the absence and presence of paclitaxel, nocodazole, or roscovitine, and the activity of endogenous Ppp4c-R2-R3A is inhibited from G1/S to the G2/M phase of the cell cycle. Endogenous γ-tubulin and its associated protein, γ-tubulin complex protein 2, both of which are essential for nucleation of microtubules at centrosomes, interact with the Ppp4 complex. Recombinant γ-tubulin can be phosphorylated by Cdk1-cyclin B or Brsk1 and dephosphorylated by Ppp4c-R2-R3A in vitro. The data indicate that Ppp4c-R2-R3A regulates microtubule organization at centrosomes during cell division in response to stress signals such as spindle toxins, paclitaxel, and nocodazole, and that inhibition of the Ppp4 complex may be advantageous for treatment of some cancers.

Glucose-6-phosphate dehydrogenase regulation in the hepatopancreas of the anoxia-tolerant marine mollusc,Littorina littorea

Glucose-6-phosphate dehydrogenase (G6PDH) gates flux through the pentose phosphate pathway and is key to cellular antioxidant defense due to its role in producing NADPH. Good antioxidant defenses are crucial for anoxia-tolerant organisms that experience wide variations in oxygen availability. The marine mollusc, Littorina littorea, is an intertidal snail that experiences daily bouts of anoxia/hypoxia with the tide cycle and shows multiple metabolic and enzymatic adaptations that support anaerobiosis. This study investigated the kinetic, physical and regulatory properties of G6PDH from hepatopancreas of L. littorea to determine if the enzyme is differentially regulated in response to anoxia, thereby providing altered pentose phosphate pathway functionality under oxygen stress conditions. Several kinetic properties of G6PDH differed significantly between aerobic and 24 h anoxic conditions; compared with the aerobic state, anoxic G6PDH (assayed at pH 8) showed a 38% decrease in Km G6P and enhanced inhibition by urea, whereas in pH 6 assays Km NADP and maximal activity changed significantly between the two states. The mechanism underlying anoxia-responsive changes in enzyme properties proved to be a change in the phosphorylation state of G6PDH. This was documented with immunoblotting using an anti-phosphoserine antibody, in vitro incubations that stimulated endogenous protein kinases versus protein phosphatases and significantly changed Km G6P, and phosphorylation of the enzyme with 32P-ATP. All these data indicated that the aerobic and anoxic forms of G6PDH were the high and low phosphate forms, respectively, and that phosphorylation state was modulated in response to selected endogenous protein kinases (PKA or PKG) and protein phosphatases (PP1 or PP2C). Anoxia-induced changes in the phosphorylation state of G6PDH may facilitate sustained or increased production of NADPH to enhance antioxidant defense during long term anaerobiosis and/or during the transition back to aerobic conditions when the reintroduction of oxygen causes a rapid increase in oxidative stress.

Insights into theIn VivoRegulation of Glutamate Dehydrogenase from the Foot Muscle of an Estivating Land Snail

Land snails, Otala lactea, survive in seasonally hot and dry environments by entering a state of aerobic torpor called estivation. During estivation, snails must prevent excessive dehydration and reorganize metabolic fuel use so as to endure prolonged periods without food. Glutamate dehydrogenase (GDH) was hypothesized to play a key role during estivation as it shuttles amino acid carbon skeletons into the Krebs cycle for energy production and is very important to urea biosynthesis (a key molecule used for water retention). Analysis of purified foot muscle GDH from control and estivating conditions revealed that estivated GDH was approximately 3-fold more active in catalyzing glutamate deamination as compared to control. This kinetic difference appears to be regulated by reversible protein phosphorylation, as indicated by ProQ Diamond phosphoprotein staining and incubations that stimulate endogenous protein kinases and phosphatases. The increased activity of the high-phosphate form of GDH seen in the estivating land snail foot muscle correlates well with the increased use of amino acids for energy and increased synthesis of urea for water retention during prolonged estivation.

An Inherent Ability of Ventricular Myocytes to Self-Organize Spontaneous RyRs Activation that Generate Synchronized Local Ca2+ Releases is Physiologically Suppressed by Constitutive Phosphodiesterase and Protein Phosphatase Activity

Stochastic, spontaneous, local RyRs activation underlies basal “Ca2+ sparks” in ventricular myocytes (VM). We have previously shown that addition of exogenous cAMP to saponin-permeabilized rabbit VM synchronizes stochastic Ca2+ sparks into rhythmic local Ca2+ releases. We hypothesized that in the basal state VM have an intrinsic ability to self-organize spontaneous RyRs activation and synchronize Ca2+ releases but this self-organization is suppressed by endogenous phosphodiesterase (PDE) and protein phosphatase (PP) activity, permitting only the rare and stochastic occurrence of Ca2+ sparks. We found that incubation of saponin-permeabilized VM (confocal imaging of Fluo-4 saltfluorescence) with a PP inhibitor (CyA 0.5 μM) or/and PDE inhibitor (IBMX 10μM) converts stochastic sparks into periodic, spatially synchronized local Ca2+ releases (Table). Moreover, the combination PP and PDE inhibitors produced rhythmic, local Ca2+ releases with a shorter period than that produced by either of inhibitors alone. Thus, VM have an inherent capacity to self-organize spontaneous basal RyR activation and synchronize Ca2+ release, but this is physiologically suppressed by constitutive PDE and PP activity.

An enzymatic bridge between carbohydrate and amino acid metabolism: regulation of glutamate dehydrogenase by reversible phosphorylation in a severe hypoxia-tolerant crayfish

Glutamate dehydrogenase (GDH) (EC 1.4.1.3) is a crucial enzyme involved in bridging two metabolic pathways, gating the use of glutamate for either amino acid metabolism, or carbohydrate metabolism. The present study investigated GDH from tail muscle of the freshwater crayfish Orconectes virilis exploring changes to kinetic properties, phosphorylation levels and structural stability between two forms of the enzyme (aerobic control and 20-h severe hypoxic). Evidence indicated that GDH was converted to a high phosphate form under oxygen limitation. ProQ Diamond phosphoprotein staining showed a 42% higher bound phosphate content on GDH from muscle of severely hypoxic crayfish compared with the aerobic form, and treatment of this GDH with commercial phosphatase (alkaline phosphatase), and treatments that stimulated the activities of different endogenous protein phosphatases (stimulating PP1+PP2A, PP2B, and PP2C) yielded significant increases in the fold activation by ADP of GDH from both control and severe hypoxic conditions. By contrast, stimulation of the activities of endogenous protein kinases (AMPK, PKA or CaMK) significantly reduced the ADP fold activation from control animals. The physiological consequence of severe hypoxia-induced GDH phosphorylation may be to suppress GDH activity under low oxygen, shutting off this critical bridge point between two metabolic pathways.

Relief of autoinhibition of the electrogenic Na-HCO3 cotransporter NBCe1-B: role of IRBIT vs. amino-terminal truncation

Two maneuvers known to stimulate electrogenic sodium bicarbonate cotransporter 1 (NBCe1) activity are 1) deletion from the cytosolic amino-terminus (Nt) of NBCe1-C of an 87-amino acid sequence that contains an autoinhibitory domain (AID); and 2) binding of the protein IRBIT to elements within the same 87-amino acid module in a different variant, NBCe1-B. Helpful to understanding the relationship between these two phenomena would be an appreciation of the relative magnitude of stimulation caused by each maneuver for the same NBCe1 variant. In the present study, we performed two-electrode voltage-clamp on Xenopus oocytes expressing human NBCe1-B constructs, with and without human IRBIT constructs. We find that removal of the AID stimulates NBCe1-B to the same extent as coexpression of wild-type IRBIT. The potency of wild-type IRBIT apparently is reduced by the action of endogenous oocyte protein phosphatases: a mutant IRBIT that cannot be influenced by the action of protein phosphatase-1 stimulates NBCe1-B to an extent 50% greater than can be achieved by removal of the NBCe1-B AID. Thus the stimulatory effect of IRBIT cannot be explained solely by masking of autoinhibitory determinants within the AID. Finally, we find that an NBCe1-B construct that lacks amino acid residues 2-16 of the Nt is fully autoinhibited, but cannot be stimulated by IRBIT, indicating that autoinhibitory and IRBIT-binding determinants within the cytosolic Nt are not identical.

Reversible phosphorylation regulation of NADPH‐linked polyol dehydrogenase in the freeze‐avoiding gall moth, Epiblema scudderiana: role in glycerol metabolism

Larvae of the goldenrod gall moth, Epiblema scudderiana, use a freeze avoidance strategy of cold hardiness to survive the winter. A key metabolic adaption that supports subzero survival is the accumulation of large amounts of glycerol as a colligative antifreeze. Production of glycerol relies on polyol dehydrogenase (PDH) which catalyzes the NADPH-dependent conversion of glyceraldehyde into glycerol. Kinetic analysis of PDH from E. scudderiana revealed significant changes in properties as a result of subzero temperature acclimation; the K(m) for glyceraldehyde in 5°C-acclimated larvae was 7.0 mM and doubled in - 15°C-exposed larvae. This change suggested that PDH is regulated by a state-dependent covalent modification. Indeed, high and low K(m) forms could be interconverted by incubating larval extracts in vitro under conditions that stimulated either endogenous protein kinases or protein phosphatases. Protein kinase incubations doubled the K(m) glyceraldehyde of the 5°C enzyme, whereas protein phosphatase incubations decreased the K(m) of the - 15°C enzyme by about 50%. PDH was purified by ion exchange and affinity chromatography steps and then subjected to electrophoresis. Staining with ProQ Diamond phosphoprotein stain showed a much higher phosphate content of PDH from - 15°C-acclimated larvae, a result that was further confirmed by immunoblotting that showed a much greater phosphoserine content on the - 15°C enzyme. These experiments established that PDH is regulated by state-dependent reversible phosphorylation in E. scudderiana and suggest that this regulatory mechanism makes a significant contribution to controlling the synthesis, maintenance, and degradation of glycerol pools over the winter months.

Multi-mechanisms are involved in reactive oxygen species regulation of mTORC1 signaling

The mammalian target of rapamycin complex 1(mTORC1) integrates diverse signals to control cell growth, proliferation, survival, and metabolism. Role of reactive oxygen species (ROS) on mTORC1 signaling remains obscure and mechanisms through which ROS modulate mTORC1 are not known. We demonstrate that low doses ROS exposure stimulate mTORC1 while high concentrations or long-term ROS treatment decrease mTORC1 activity in vivo and in a variety of cell lines. The dose/time needed for inhibition or activation are cell type-dependent. In HEK293 cells hydrogen peroxide (H2O2) stimulates phosphorylation of AMP-activated kinase (AMPK) (T172) and Raptor (S792), enhances association of activated AMPK with Raptor. Furthermore, AMPK inhibitor compound c inhibits H2O2-induced Raptor (S792) phosphorylation and reverses H2O2-induced de-phosphorylation of mTORC1 downstream targets p70-S6K1 (T389), S6 (S235/236) and 4E-BP1 (T37/46). H2O2 also stimulates association of endogenous protein phosphatase 2A catalytic subunit (PP2Ac) with p70-S6K1. Like compound c, inhibitor of PP2A, okadaic acid partially reverses inactivation of mTORC1 substrates induced by H2O2. Moreover, inhibition of PP2A and AMPK partially rescued cells from H2O2-induced cell death. High doses of H2O2 inhibit while low doses of H2O2 activate mTORC1 both in TSC2−/− P53−/− and TSC2+/+ P53−/− MEFs. These data suggest that PP2A and AMPK-mediated phosphorylation of Raptor mediate H2O2-induced inhibition of mTORC1 signaling.

In Xenopus oocytes, stimulation of the electrogenic Na/HCO3 transporter NBCe1 by IRBIT can be explained by relief of transporter autoinhibition and is unaffected by endogenous phosphatases.

Other have shown in Xenopus oocytes that NBCe1 lacking an amino‐terminal (Nt) autoinhibitory sequence has a ~3‐fold greater transport activity than full‐length NBCe1. In a separate study, it was reported that co‐expression of full‐length NBCe1 with its Nt binding partner IRBIT results in a ~7‐fold stimulation of transporter activity. At face value these data suggest that the stimulatory action of IRBIT cannot be explained solely by relief of autoinhibition. However, these data pertain to different splice variants of NBCe1.In the present study, we work only with the pancreatic splice form NBCe1‐B. We find that, compared to oocytes expressing full‐length NBCe1‐B alone, oocytes expressing (1) a mutant NBCe1‐B that lacks the autoinhibitory sequence manifest a 7.5‐fold greater transporter activity (2) full‐length NBCe1‐B plus IRBIT manifest a 5.2‐fold greater transporter activity.Oocytes have endogenous protein phosphatase 1 (PP1) and PP1 is reported to deactivate IRBIT following binding at a non‐consensus PP1‐binding site ‘KQIQF’ in IRBIT. Thus we hypothesized that the full stimulatory effect of IRBIT was masked by the action of endogenous PP1. However, we find that removing the putative PP1 binding site in IRBIT does not result in a substantial increase in NBCe1‐B stimulation. Conversely an IRBIT in which we replaced ‘KQIQF’ with a well characterized PP1‐binding consensus sequence of the type ‘RVxF’ was unable to stimulate NBCe1‐B activity.In conclusion, our findings are consistent with a model in which the action of IRBIT is explained solely by relief of autoinhibition. The results are not confounded by endogenous oocyte PP1 activity, because the endogenous PP1 has no detectable functional interaction with the ‘KQIQF’ motif in overexpressed IRBIT.

Deactivation of Sphingosine Kinase 1 by Protein Phosphatase 2A

Sphingosine kinase 1 (SK1) is an important regulator of cellular signaling that has been implicated in a broad range of cellular processes. Cell exposure to a wide array of growth factors, cytokines, and other cell agonists can result in a rapid and transient increase in SK activity via an activating phosphorylation. We have previously identified extracellular signal-regulated kinases 1 and 2 (ERK1/2) as the kinases responsible for the phosphorylation of human SK1 at Ser(225), but the corresponding phosphatase targeting this phosphorylation has remained undefined. Here, we provide data to support a role for protein phosphatase 2A (PP2A) in the deactivation of SK1 through dephosphorylation of phospho-Ser(225). The catalytic subunit of PP2A (PP2Ac) was found to interact with SK1 using both GST-pulldown and coimmunoprecipitation analyses. Coexpression of PP2Ac with SK1 resulted in reduced Ser(225) phosphorylation of SK1 in human embryonic kidney (HEK293) cells. In vitro phosphatase assays showed that PP2Ac dephosphorylated both recombinant SK1 and a phosphopeptide based on the phospho-Ser(225) region of SK1. Finally, both basal and tumor necrosis factor-alpha-stimulated cellular SK1 activity were regulated by molecular manipulation of PP2Ac activity. Thus, PP2A appears to function as an endogenous regulator of SK1 phosphorylation.

Regulation of skeletal muscle creatine kinase from a hibernating mammal

Control over skeletal muscle energetics is critical in hibernation to sustain viability over weeks of cold torpor and to support shivering thermogenesis during arousal. Creatine kinase (CK)1Abbreviations used: CK, creatine kinase; Cr, creatine; PCr, phosphocreatine; PKC, protein kinase C.1 has a key role in muscle energetics and this study analyzes muscle CK from ground squirrels, Spermophilus richardsonii. CK activity was ∼20% lower during hibernation than in euthermia, as was CK protein whereas CK mRNA was reduced by ∼70%. Hibernator CK showed reduced affinity for ATP and creatine, compared with euthermic CK. Incubations that promoted endogenous protein kinase or phosphatase action, coupled with ion exchange chromatography to separate high and low phosphate forms, showed that soluble CK from euthermic squirrels was a mix of phosphorylated and dephosphorylated forms whereas only phospho-CK was detected in hibernating animals. High and low phosphate CK forms had different affinities for ATP and creatine substrates but did not differ in stability to urea denaturation. About 20–25% of CK was bound to the insoluble fraction of muscle and bound CK showed different kinetic responses to kinase and phosphatase treatments.