Skeleton binding protein 1 localizes to the Maurer's cleft and interacts with PfHSP70-1 and PfHSP70-x in Plasmodium falciparum gametocyte-infected erythrocytes.

Plasmodium falciparum accounts for the majority of malaria deaths, due to pathology provoked by the ability of infected erythrocytes to adhere to vascular endothelium within deep tissues. The parasite recognizes endothelium by trafficking and displaying protein ligands on the surface of asexual stage infected erythrocytes, such as members of the large family of pathogenic proteins, P. falciparum erythrocyte membrane protein 1 (PfEMP1). Parasite-encoded skeleton binding protein 1 (SBP1) plays an important role in the transport of these binding-related surface proteins, via cleft-like membranous structures termed Maurer's clefts, which are present within the cytoplasm of infected erythrocytes. Erythrocytes infected with gametocyte stages accumulate in the extravascular compartment of bone marrow; and it was suggested that their surface-expressed adhesion molecule profile and protein trafficking mechanisms might differ from those in asexual stage parasites. Protein trafficking mechanisms via Maurer's clefts have been well investigated in asexual stage parasite-infected erythrocytes; but little is known regarding the gametocyte stages. In this study, we characterized SBP1 during gametocyte maturation and demonstrated that SBP1 is expressed and localizes to dot-like Maurer's cleft structures in the cytoplasm of gametocyte-infected erythrocytes. Co-immunoprecipitation and mass spectrometry assays indicated that SBP1 interacts with the molecular chaperones PfHSP70-1 and PfHSP70-x. Localization analysis suggested that some PfHSP70-1 and/or PfHSP70-x localize in a dot-like pattern within the cytoplasm of immature gametocyte-infected erythrocytes. These findings suggest that SBP1 may interact with HSP70 chaperones in the infected erythrocyte cytoplasm during the immature gametocyte stages.

Polo-Like Kinase 2 Plays an Essential Role in Cytoprotection against MG132-Induced Proteasome Inhibition via Phosphorylation of Serine 19 in HSPB5.

Protein homeostasis, including protein folding, refolding, and degradation, is thought to decline with aging. HSPB5 (also known as αB-crystallin) prevents target protein aggregation as a molecular chaperone and exhibits a cytoprotective function against various cell stresses. To elucidate the effect of HSPB5 on endoplasmic reticulum (ER) stress, we searched for novel binding proteins of HSPB5 using the proximity-dependent biotin labeling method. Proteins presumed to interact with HSPB5 in cells treated with the proteasome inhibitor MG132 were identified by a reversible biotin-binding capacity method combining tamavidin2-REV magnetic beads and mass spectrometry. We discovered a new binding protein for HSPB5, polo-like kinase 2 (PLK2), which is an apoptosis-related enzyme. The expression of PLK2 was upregulated by MG132 treatment, and it was co-localized with HSPB5 near the ER in L6 muscle cells. Inhibition of PLK2 decreased ER stress-induced phosphorylation of serine 19 in HSPB5 and increased apoptosis by activation of caspase 3 under ER stress. Overexpression of HSPB5 (WT) suppressed the ER stress-induced caspase 3 activity, but this was not observed with phospho-deficient HSPB5 (3A) mutants. These results clarify the role of HSPB5 phosphorylation during ER stress and suggest that the PLK2/HSPB5 pathway plays an essential role in cytoprotection against proteasome inhibition-induced ER stress.

Gene Expression Analysis Provides New Insights into the Mechanism of Intramuscular Fat Formation in Japanese Black Cattle.

Japanese Black cattle (Japanese Wagyu) have a unique phenotype in which ectopic intramuscular fat accumulates in skeletal muscle, producing finely marbled beef. However, the mechanism of intramuscular fat formation in Japanese Black cattle remains unclear. To investigate the key genes involved in intramuscular fat accumulation, we comprehensively analyzed mRNA levels in subcutaneous and intramuscular fat tissues using RNA sequence (RNA-seq) analysis, which detected 27,606 genes. We identified eight key genes, namely carboxypeptidase E, tenascin C, transgelin, collagen type IV alpha 5 (COL4A5), cysteine and glycine-rich protein 2, PDZ, and LIM domain 3, phosphatase 1 regulatory inhibitor subunit 14A, and regulator of calcineurin 2. These genes were highly and specifically expressed in intramuscular fat tissue. Immunohistochemical analysis revealed a collagen network, including COL4A5, in the basement membrane around the intramuscular fat tissue. Moreover, pathway analysis revealed that, in intramuscular fat tissue, differentially expressed genes are related to cell adhesion, proliferation, and cancer pathways. Furthermore, pathway analysis showed that the transforming growth factor-ß (TGF-ß) and small GTPases regulators RASGRP3, ARHGEF26, ARHGAP10, ARHGAP24, and DLC were upregulated in intramuscular fat. Our study suggests that these genes are involved in intramuscular fat formation in Japanese Black cattle.

The C-terminal flexible region of branched-chain polyamine synthase facilitates substrate specificity and catalysis.

Branched-chain polyamine synthase (BpsA) catalyzes sequential aminopropyl transfer from the donor, decarboxylated S-adenosylmethionine (dcSAM), to the acceptor, linear-chain polyamine, resulting in the production of a quaternary-branched polyamine via tertiary branched polyamine intermediates. Here, we analyzed the catalytic properties and X-ray crystal structure of Tth-BpsA from Thermus thermophilus and compared them with those of Tk-BpsA from Thermococcus kodakarensis, which revealed differences in acceptor substrate specificity and C-terminal structure between these two enzymes. To investigate the role of the C-terminal flexible region in acceptor recognition, a region (QDEEATTY) in Tth-BpsA was replaced with that in Tk-BpsA (YDDEESSTT) to create chimeric Tth-BpsA C9, which showed a severe reduction in catalytic efficiency toward N4 -aminopropylnorspermidine, but not toward N4 -aminopropylspermidine, mimicking Tk-BpsA substrate specificity. Tth-BpsA C9 Tyr346 and Thr354 contributed to discrimination between tertiary branched-chain polyamine substrates, suggesting that the C-terminal region of BpsA recognizes acceptor substrates. Liquid chromatography-tandem mass spectrometry analysis on a Tk-BpsA reaction mixture with dcSAM revealed two aminopropyl groups bound to two of five aspartate/glutamate residues (Glu339 , Asp342 , Asp343 , Glu344 , and Glu345 ) in the C-terminal flexible region. Mutating each of these five amino acid residues to asparagine/glutamine resulted in a slight decrease in activity. The quadruple mutant D342N/D343N/E344Q/E345Q exhibited a severe reduction in catalytic efficiency, suggesting that these aspartate/glutamate residues function to receive aminopropyl chains. In addition, the X-ray crystal structure of the Tk-BpsA ternary complex bound to N4 -bis(aminopropyl)spermidine revealed that Asp126 and Glu259 interacted with the aminopropyl moiety in N4 -aminopropylspermidine.

The Role of Cysteine String Protein α Phosphorylation at Serine 10 and 34 by Protein Kinase Cγ for Presynaptic Maintenance.

Protein kinase Cγ (PKCγ) knock-out (KO) animals exhibit symptoms of Parkinson's disease (PD), including dopaminergic neuronal loss in the substantia nigra. However, the PKCγ substrates responsible for the survival of dopaminergic neurons in vivo have not yet been elucidated. Previously, we found 10 potent substrates in the striatum of PKCγ-KO mice. Here, we focused on cysteine string protein α (CSPα), a protein from the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles. We found that in cultured cells, PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. Additionally, apoptosis was found to have been enhanced by the overexpression of a phosphorylation-null mutant of CSPα, CSPα(S10A/S34A). Compared with wild-type (WT) CSPα, the CSPα(S10A/S34A) mutant had a weaker interaction with HSP70. However, in sharp contrast, a phosphomimetic CSPα(S10D/S34D) mutant, compared with WT CSPα, had a stronger interaction with HSP70. In addition, total levels of synaptosomal-associated protein (SNAP) 25, a main downstream target of the HSC70/HSP70 chaperone complex, were found to have decreased by the CSPα(S10A/S34A) mutant through increased ubiquitination of SNAP25 in PC12 cells. In the striatum of 2-year-old male PKCγ-KO mice, decreased phosphorylation levels of CSPα and decreased SNAP25 protein levels were observed. These findings indicate the phosphorylation of CSPα by PKCγ may protect the presynaptic terminal from neurodegeneration. The PKCγ-CSPα-HSC70/HSP70-SNAP25 axis, because of its role in protecting the presynaptic terminal, may provide a new therapeutic target for the treatment of PD.SIGNIFICANCE STATEMENT Cysteine string protein α (CSPα) is a protein belonging to the heat shock protein (HSP) 40 cochaperone families localized on synaptic vesicles, which maintain the presynaptic terminal. However, the function of CSPα phosphorylation by protein kinase C (PKC) for neuronal cell survival remains unclear. The experiments presented here demonstrate that PKCγ phosphorylates CSPα at serine (Ser) 10 and Ser34. CSPα phosphorylation at Ser10 and Ser34 by PKCγ protects the presynaptic terminal by promoting HSP70 chaperone activity. This report suggests that CSPα phosphorylation, because of its role in modulating HSP70 chaperone activity, may be a target for the treatment of neurodegeneration.

Overexpression of eIF5 or its protein mimic 5MP perturbs eIF2 function and induces ATF4 translation through delayed re-initiation.

ATF4 is a pro-oncogenic transcription factor whose translation is activated by eIF2 phosphorylation through delayed re-initiation involving two uORFs in the mRNA leader. However, in yeast, the effect of eIF2 phosphorylation can be mimicked by eIF5 overexpression, which turns eIF5 into translational inhibitor, thereby promoting translation of GCN4, the yeast ATF4 equivalent. Furthermore, regulatory protein termed eIF5-mimic protein (5MP) can bind eIF2 and inhibit general translation. Here, we show that 5MP1 overexpression in human cells leads to strong formation of 5MP1:eIF2 complex, nearly comparable to that of eIF5:eIF2 complex produced by eIF5 overexpression. Overexpression of eIF5, 5MP1 and 5MP2, the second human paralog, promotes ATF4 expression in certain types of human cells including fibrosarcoma. 5MP overexpression also induces ATF4 expression in Drosophila The knockdown of 5MP1 in fibrosarcoma attenuates ATF4 expression and its tumor formation on nude mice. Since 5MP2 is overproduced in salivary mucoepidermoid carcinoma, we propose that overexpression of eIF5 and 5MP induces translation of ATF4 and potentially other genes with uORFs in their mRNA leaders through delayed re-initiation, thereby enhancing the survival of normal and cancer cells under stress conditions.

Hepatitis C virus NS5A protein interacts with lysine methyltransferase SET and MYND domain-containing 3 and induces activator protein 1 activation.

Hepatitis C virus (HCV) non-structural protein 5A (NS5A) is a multifunctional protein that is involved in the HCV life cycle and pathogenesis. In this study, a host protein(s) interacting with NS5A by tandem affinity purification were searched for with the aim of elucidating the role of NS5A. An NS5A-interacting protein, SET and MYND domain-containing 3 (SMYD3), a lysine methyltransferase reportedly involved in the development of cancer, was identified. The interaction between NS5A and SMYD3 was confirmed in ectopically expressing, HCV RNA replicon-harboring and HCV-infected cells. The other HCV proteins did not bind to SMYD3. SMYD3 bound to NS5A of HCV genotypes 1b and 2a. Deletion mutational analysis revealed that domains II and III of NS5A (amino acids [aa] 250 to 447) and the MYND and N-SET domains of SMYD3 (aa 1 to 87) are involved in the full extent of NS5A-SMYD3 interaction. NS5A co-localized with SMYD3 exclusively in the cytoplasm, thereby inhibiting nuclear localization of SMYD3. Moreover, NS5A formed a complex with SMYD3 and heat shock protein 90 (HSP90), which is a positive regulator of SMYD3. The intensity of binding between SMYD3 and HSP90 was enhanced by NS5A. Luciferase reporter assay demonstrated that NS5A significantly induces activator protein 1 (AP-1) activity, this being potentiated by co-expression of SMYD3 with NS5A. Taken together, the present results suggest that NS5A interacts with SMYD3 and induces AP-1 activation, possibly by facilitating binding between HSP90 and SMYD3. This may be a novel mechanism of AP-1 activation in HCV-infected cells.

Chicken heat shock protein HSPB1 increases and interacts with αB-crystallin in aged skeletal muscle.

International trading markets of meat require the animal's age information to prevent cross-contamination of ineligible meat products. Individual livestock age is either evaluated from physiological features or verified by breeding history. However, it remains impossible to perform age verification on meat when a suspicion of error occurred in the importing country. To investigate an age-related protein in skeletal muscle of livestock, we compared protein expression among chicken pectoralis major of different ages. Results indicated that the level of expression of chicken HSPB1, one of the small heat shock proteins, was increased in aged muscles. On the other hand, other heat shock proteins, heat shock factors, and myosin heavy chain isoform did not change the expression levels in aged chicken muscle. In addition, we identified that αB-crystallin interacted with HSPB1 in aged chicken muscle. These results suggest that HSPB1 protein forms complexes with αB-crystallin in aged chicken muscle and suppose to become the candidate of age-related bio-marker for verifying the age of chicken meat.

Functional regulation of the DNA damage-recognition factor DDB2 by ubiquitination and interaction with xeroderma pigmentosum group C protein.

In mammalian nucleotide excision repair, the DDB1-DDB2 complex recognizes UV-induced DNA photolesions and facilitates recruitment of the XPC complex. Upon binding to damaged DNA, the Cullin 4 ubiquitin ligase associated with DDB1-DDB2 is activated and ubiquitinates DDB2 and XPC. The structurally disordered N-terminal tail of DDB2 contains seven lysines identified as major sites for ubiquitination that target the protein for proteasomal degradation; however, the precise biological functions of these modifications remained unknown. By exogenous expression of mutant DDB2 proteins in normal human fibroblasts, here we show that the N-terminal tail of DDB2 is involved in regulation of cellular responses to UV. By striking contrast with behaviors of exogenous DDB2, the endogenous DDB2 protein was stabilized even after UV irradiation as a function of the XPC expression level. Furthermore, XPC competitively suppressed ubiquitination of DDB2 in vitro, and this effect was significantly promoted by centrin-2, which augments the DNA damage-recognition activity of XPC. Based on these findings, we propose that in cells exposed to UV, DDB2 is protected by XPC from ubiquitination and degradation in a stochastic manner; thus XPC allows DDB2 to initiate multiple rounds of repair events, thereby contributing to the persistence of cellular DNA repair capacity.

The role of Pak-interacting exchange factor-ß phosphorylation at serines 340 and 583 by PKCγ in dopamine release.

Protein kinase C (PKC) has been implicated in the control of neurotransmitter release. The AS/AGU rat, which has a nonsense mutation in PKCγ, shows symptoms of parkinsonian syndrome, including dopamine release impairments in the striatum. Here, we found that the AS/AGU rat is PKCγ-knock-out (KO) and that PKCγ-KO mice showed parkinsonian syndrome. However, the PKCγ substrates responsible for the regulated exocytosis of dopamine in vivo have not yet been elucidated. To identify the PKCγ substrates involved in dopamine release, we used PKCγ-KO mice and a phosphoproteome analysis. We found 10 candidate phosphoproteins that had decreased phosphorylation levels in the striatum of PKCγ-KO mice. We focused on Pak-interacting exchange factor-ß (ßPIX), a Cdc42/Rac1 guanine nucleotide exchange factor, and found that PKCγ directly phosphorylates ßPIX at Ser583 and indirectly at Ser340 in cells. Furthermore, we found that PKC phosphorylated ßPIX in vivo. Classical PKC inhibitors and ßPIX knock-down (KD) significantly suppressed Ca(2+)-evoked dopamine release in PC12 cells. Wild-type ßPIX, and not the ßPIX mutants Ser340 Ala or Ser583 Ala, fully rescued the decreased dopamine release by ßPIX KD. Double KD of Cdc42 and Rac1 decreased dopamine release from PC12 cells. These findings indicate that the phosphorylation of ßPIX at Ser340 and Ser583 has pivotal roles in Ca(2+)-evoked dopamine release in the striatum. Therefore, we propose that PKCγ positively modulates dopamine release through ß2PIX phosphorylation. The PKCγ-ßPIX-Cdc42/Rac1 phosphorylation axis may provide a new therapeutic target for the treatment of parkinsonian syndrome.

Association of CAD, a multifunctional protein involved in pyrimidine synthesis, with mLST8, a component of the mTOR complexes.

BACKGROUND: mTOR is a genetically conserved serine/threonine protein kinase, which controls cell growth, proliferation, and survival. A multifunctional protein CAD, catalyzing the initial three steps in de novo pyrimidine synthesis, is regulated by the phosphorylation reaction with different protein kinases, but the relationship with mTOR protein kinase has not been known. RESULTS: CAD was recovered as a binding protein with mLST8, a component of the mTOR complexes, from HEK293 cells transfected with the FLAG-mLST8 vector. Association of these two proteins was confirmed by the co-immuoprecipitaiton followed by immunoblot analysis of transfected myc-CAD and FLAG-mLST8 as well as that of the endogenous proteins in the cells. Analysis using mutant constructs suggested that CAD has more than one region for the binding with mLST8, and that mLST8 recognizes CAD and mTOR in distinct ways. The CAD enzymatic activity decreased in the cells depleted of amino acids and serum, in which the mTOR activity is suppressed. CONCLUSION: The results obtained indicate that mLST8 bridges between CAD and mTOR, and plays a role in the signaling mechanism where CAD is regulated in the mTOR pathway through the association with mLST8.

Novel PKCα-mediated phosphorylation site(s) on cofilin and their potential role in terminating histamine release.

Using specific inhibitors, kinase-negative mutants, and small interfering RNA against protein kinase Cα (PKCα) or PKCßI, we find that PKCßI positively regulates degranulation in rat basophilic leukemia-2H3 cells, whereas PKCα negatively regulates degranulation. Mass spectrometric and mutagenic analyses reveal that PKCα phosphorylates cofilin at Ser-23 and/or Ser-24 during degranulation. Overexpression of a nonphosphorylatable form (S23,24A), but not that of a mutant-mimicking phosphorylated form (S23,24E), increases degranulation. Furthermore, the S23,24A mutant binds to F-actin and retains its depolymerizing and/or cleavage activity; conversely, the S23,24E mutant is unable to sever actin filaments, resulting in F-actin polymerization. In addition, the S23,24E mutant preferentially binds to the 14-3-3ζ protein. Fluorescence-activated cell sorting analysis with fluorescein isothiocyanate-phalloidin and simultaneous observation of degranulation, PKC translocation, and actin polymerization reveals that during degranulation, actin polymerization is dependent on PKCα activity. These results indicate that a novel PKCα-mediated phosphorylation event regulates cofilin by inhibiting its ability to depolymerize F-actin and bind to 14-3-3ζ, thereby promoting F-actin polymerization, which is necessary for cessation of degranulation.

c-Abl tyrosine kinase regulates serum-induced nuclear export of diacylglycerol kinase α by phosphorylation at Tyr-218.

c-Abl is a tyrosine kinase involved in many cellular processes, including cell cycle control and proliferation. However, little is known about its substrates. Here, we show that c-Abl directly phosphorylates diacylglycerol kinase α (DGKα), an important regulator of many cellular events through its conversion of diacylglycerol to phosphatidic acid. We found that DGKα was transported from the cytoplasm to the nucleus in response to serum starvation, and serum restoration induced the nuclear export of the enzyme to the cytoplasm. This serum-induced export involves two tyrosine kinases, c-Src and c-Abl. The latter, c-Abl, is activated by c-Src, phosphorylates DGKα, and shuttles between the nucleus and the cytoplasm in a direction opposite to that of DGKα in response to serum restoration. Moreover, an in vitro phosphorylation assay using purified mutants of DGKα identified Tyr-218 as a site of phosphorylation by c-Abl. We confirmed these results for endogenous DGKα using an antibody specific for phospho-Tyr-218, and this phosphorylation was necessary for the serum-induced export of DGKα. These results demonstrate that the nucleo-cytoplasmic shuttling of DGKα is orchestrated by tyrosine phosphorylation by the Src-activated tyrosine kinase c-Abl and that this phosphorylation is important for regulating the function of cytoplasmic and/or nuclear DGKα.

Direct binding of RalA to PKCη and its crucial role in morphological change during keratinocyte differentiation.

During differentiation, keratinocytes undergo a dramatic shape change from small and round to large and flat, in addition to production of proteins necessary for the formation of epidermis. It has been shown that protein kinase C (PKC) η is crucial for keratinocyte differentiation. However, its role in this process has yet to be fully elucidated. Here, we show that catalytic activity is not necessary for enlarged and flattened morphology of human keratinocytes induced by overexpression of PKCη, although it is important for gene expression of the marker proteins. In addition, we identify the small G protein RalA as a binding partner of PKCη, which binds to the C1 domain, an indispensable region for the morphological change. The binding led activation of RalA and actin depolymerization associated with keratinocyte differentiation. siRNA techniques proved that RalA is involved in not only the keratinocyte differentiation induced by PKCη overexpression but also normal keratinocyte differentiation induced by calcium and cholesterol sulfate. These results provide a new insight into the molecular mechanism of cytoskeletal regulation leading to drastic change of cell shape.

Tor directly controls the Atg1 kinase complex to regulate autophagy.

Autophagy is a bulk proteolytic process that is indispensable for cell survival during starvation. Autophagy is induced by nutrient deprivation via inactivation of the rapamycin-sensitive Tor complex1 (TORC1), a protein kinase complex regulating cell growth in response to nutrient conditions. However, the mechanism by which TORC1 controls autophagy and the direct target of TORC1 activity remain unclear. Atg13 is an essential regulatory component of autophagy upstream of the Atg1 kinase complex, and here we show that yeast TORC1 directly phosphorylates Atg13 at multiple Ser residues. Additionally, expression of an unphosphorylatable Atg13 mutant bypasses the TORC1 pathway to induce autophagy through activation of Atg1 in cells growing under nutrient-rich conditions. Our findings suggest that the direct control of the Atg1 complex by TORC1 induces autophagy.

Precursor ion scanning and sequencing of arginine-ADP-ribosylated peptide by mass spectrometry.

Arginine (Arg)-specific ADP-ribosylation is one of the posttranslational modifications of proteins and is thought to play an important role in reversibly regulating functions of the target proteins in eukaryotes. However, the physiological target protein has not been established. We examined the fragmentation pattern of both ADP-ribosyl-Arg (ADP-R-Arg) and Arg-ADP-ribosylated peptides by quadrupole tandem mass spectrometry and found a specific cleavage of ADP-R-Arg into N-(ADP-ribosyl)-carbodiimide (ADP-R-carbodiimide) and ornithine. Based on this specific fragmentation pattern, we successfully identified the modification site and sequence of Arg-ADP-ribosylated peptide using a two-step collision and showed that ADP-R-carbodiimide is an excellent marker ion for precursor ion scanning of Arg-ADP-ribosylated peptide. We propose that a combination of the precursor ion scanning with ADP-R-carbodiimide as a marker ion and two-step collision is useful in searching for physiological target proteins of Arg-ADP-ribosylation.

AMP-activated protein kinase phosphorylates glutamine : fructose-6-phosphate amidotransferase 1 at Ser243 to modulate its enzymatic activity.

Glutamine : fructose-6-phosphate amidotransferase 1 (GFAT1) was identified as a protein phosphorylated in glucose-deprived cells by immunoprecipitation using the anti-phospho Akt substrates (PAS) antibody, which recognizes the phosphorylation motif site by AMP-activated protein kinase (AMPK), followed by mass fingerprinting analysis. Glucose depletion-induced phosphorylation of endogenous GFAT was potentiated by 2-deoxyglucose (2-DG), an AMPK activator, and the 2-DG-stimulated phosphorylation of FLAG-tagged GFAT1 in transfected cells was suppressed by Compound C, an AMPK inhibitor. The 2-DG induced phosphorylation of GFAT1 was attenuated by the introduction of the kinase-negative mutant of AMPK, and the phosphorylation was observed in the cells expressing the constitutively active mutant of AMPK even in the absence of 2-DG. Subsequent analysis revealed that the PAS antibody recognized GFAT1 phosphorylated at Ser243, which is conserved among different species. The assay of the GFAT enzymatic activity in the cell lysates indicated that the 2-DG-treatment inhibited the enzymatic activity, and Compound C-preincubation partially prevented the 2-DG-induced decrease of the activity. Furthermore, the mutant replacing Ser243 by alanine partially prevented the decrease of GFAT activity by 2-DG treatment. These results indicate that the phosphorylation of GFAT1 at Ser243 by AMPK has an important role in the regulation of the GFAT1 enzymatic activity.

AMP-activated protein kinase phosphorylates Golgi-specific brefeldin A resistance factor 1 at Thr1337 to induce disassembly of Golgi apparatus.

Sufficiency and depletion of nutrients regulate the cellular activities through the protein phosphorylation reaction; however, many protein substrates remain to be clarified. GBF1 (Golgi-specific brefeldin A resistance factor 1), a guanine nucleotide exchange factor for the ADP-ribosylation factor family associated with the Golgi apparatus, was isolated as a phosphoprotein from the glucose-depleted cells by using the phospho-Akt-substrate antibody, which recognizes the substrate proteins of several protein kinases. The phosphorylation of GBF1 was induced by 2-deoxyglucose (2-DG), which blocks glucose utilization and increases the intracellular AMP concentration, and by AICAR, an AMP-activated protein kinase (AMPK) activator. This phosphorylation was observed in the cells expressing the constitutively active AMPK. The 2-DG-induced phosphorylation of GBF1 was suppressed by Compound C, an AMPK inhibitor, and by the overexpression of the kinase-negative AMPK. Analysis using the deletion and point mutants identified Thr(1337) as the 2-DG-induced phosphorylation site in GBF1, which is phosphorylated by AMPK in vitro. ATP depletion is known to provoke the Golgi apparatus disassembly. Immunofluorescent microscopic analysis with the Golgi markers indicated that GBF1 associates with the fragmented Golgi apparatus in the cells treated with 2-DG and AICAR. The expression of the kinase-negative AMPK and the GBF1 mutant replacing Thr(1337) by Ala prevented the 2-DG-induced Golgi disassembly. These results indicate that GBF1 is a novel AMPK substrate and that the AMPK-mediated phosphorylation of GBF1 at Thr(1337) has a critical role, presumably by attenuating the function of GBF1, in the disassembly of the Golgi apparatus induced under stress conditions that lower the intracellular ATP concentration.

Identification of TBC7 having TBC domain as a novel binding protein to TSC1-TSC2 complex.

TBC7, a TBC (Tre-2/Bub2/Cdc16) 1 domain protein, was identified as a novel binding protein to the TSC1-TSC2 tumor suppressor complex by peptide mass fingerprinting analysis of the proteins immunoprecipitated with FLAG-epitope tagged TSC1 and TSC2 from the transfected mammalian cells. The in vivo and in vitro association of TBC7 and the TSC1-TSC2 complex was confirmed by the co-immunoprecipitation and pull-down analysis, respectively, and TBC7 was revealed to bind to the C-terminal half region of TSC1, which is distinct from the binding site with TSC2. The immunofluorescence microscopy and subcellular fractionation showed that TBC7 co-localizes with the tumor suppressor complex in the endomembrane. Overexpression of TBC7 enhanced ubiquitination of TSC1 and increased phosphorylation of S6 protein by S6 kinase, that is located in the mTOR-signaling pathway. These results indicate TBC7 could take a part in the negative regulation of the tumor suppressor complex through facilitating the downregulation of TSC1.

The proline-rich Akt substrate of 40 kDa (PRAS40) is a physiological substrate of mammalian target of rapamycin complex 1.

The proline-rich Akt substrate of 40 kilodaltons (PRAS40) was identified as a raptor-binding protein that is phosphorylated directly by mammalian target of rapamycin (mTOR) complex 1 (mTORC1) but not mTORC2 in vitro, predominantly at PRAS40 (Ser(183)). The binding of S6K1 and 4E-BP1 to raptor requires a TOR signaling (TOS) motif, which contains an essential Phe followed by four alternating acidic and small hydrophobic amino acids. PRAS40 binding to raptor was severely inhibited by mutation of PRAS40 (Phe(129) to Ala). Immediately carboxyl-terminal to Phe(129) are two small hydrophobic amino acid followed by two acidic residues. PRAS40 binding to raptor was also abolished by mutation of the major mTORC1 phosphorylation site, Ser(183), to Asp. PRAS40 (Ser(183)) was phosphorylated in intact cells; this phosphorylation was inhibited by rapamycin, by 2-deoxyglucose, and by overexpression of the tuberous sclerosis complex heterodimer. PRAS40 (Ser(183)) phosphorylation was also inhibited reversibly by withdrawal of all or of only the branched chain amino acids; this inhibition was reversed by overexpression of the Rheb GTPase. Overexpressed PRAS40 suppressed the phosphorylation of S6K1 and 4E-BP1 at their rapamycin-sensitive phosphorylation sites, and reciprocally, overexpression of S6K1 or 4E-BP1 suppressed phosphorylation of PRAS40 (Ser(183)) and its binding to raptor. RNA interference-induced depletion of PRAS40 enhanced the amino acid-stimulated phosphorylation of both S6K1 and 4E-BP1. These results establish PRAS40 as a physiological mTORC1 substrate that contains a variant TOS motif. Moreover, they indicate that the ability of raptor to bind endogenous substrates is limiting for the activity of mTORC1 in vivo and is therefore a potential locus of regulation.