Phosphorylation and Up-regulation of Diacylglycerol Kinase γ via Its Interaction with Protein Kinase Cγ

Yamaguchi, Yasushi; Shirai, Yasuhito; Matsubara, Takehiro; Sanse, Koichi; Kuriyama, Masami; Oshiro, Noriko; Yoshino, Ken‐ichi; Yonezawa, Kazuyoshi; Ono, Yoshitaka; Saito, Naoaki

doi:10.1074/jbc.m606992200

Cited by 39 publications

(46 citation statements)

References 45 publications

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“…Mass Spectrometry-The analysis was carried out as described previously (28,29). Briefly, FLAG-raptor immunoprecipitates were separated by SDS-PAGE and visualized by silver staining.…”

Section: Methodsmentioning

confidence: 99%

The Proline-rich Akt Substrate of 40 kDa (PRAS40) Is a Physiological Substrate of Mammalian Target of Rapamycin Complex 1

Oshiro

Takahashi

Yoshino

et al. 2007

Journal of Biological Chemistry

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287

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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.The mammalian target of rapamycin (mTOR) 3 is the founding member of the PI-3 kinase-related family of protein (Ser/ Thr) kinases (PIKKs) and controls many important aspects of the cellular response to nutrient sufficiency and growth factors (1). The mTOR polypeptide is now known to function in two distinct, independently regulated hetero-oligomeric complexes, called mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). Both complexes contain mTOR and the polypeptide mLST8/G␤L; mTORC1 in addition contains raptor (an ortholog of Saccharomyces cerevisiae KOG1), which binds directly to the known mTORC1 substrates S6K1 and 4E-BP1 and is indispensable for their phosphorylation by mTOR in vivo and in vitro. mTORC2 lacks raptor but contains the polypeptides rictor (an ortholog to ScAVO3) and mSin1 (an ortholog of ScAVO1). mTORC2 is one of the activating kinases for Akt, previously called PDK2, and also regulates the actin cytoskeleton through as yet unidentified effectors. Although rapamycin, in complex with FKBP12, binds directly to mTOR in a segment just amino-terminal to the catalytic domain, only mTORC1 binds the FKBP12-rapamycin complex, and thus only mTORC1 is directly susceptible to inhibition by rapamycin.Rapamycin is among the most selective kinase inhibitors k...

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“…Mass Spectrometry-The analysis was carried out as described previously (28,29). Briefly, FLAG-raptor immunoprecipitates were separated by SDS-PAGE and visualized by silver staining.…”

Section: Methodsmentioning

confidence: 99%

The Proline-rich Akt Substrate of 40 kDa (PRAS40) Is a Physiological Substrate of Mammalian Target of Rapamycin Complex 1

Oshiro

Takahashi

Yoshino

et al. 2007

Journal of Biological Chemistry

Self Cite

287

301

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“…One pathway for DAG metabolization is phosphorylation of the free hydroxyl group to produce PA. DAG is phosphorylated by DAGKs to produce PA, lowering the level of DAG and consequently downregulating PKC activation. Direct interaction between DAGKs and PKCs, DAGK ␥ association with PKC ␥ ( 192 ), DAGK association with PKC ( 193 ), and DAGK association with PKC ␣ ( 194 ) have also been reported. DAGK activity was fi rst observed in rat brain by Hokin and Hokin ( 195 ) and its activity has been detected in various subcellular compartments in a wide range of tissues and cells.…”

Section: Dagkmentioning

confidence: 99%

Lipid second messengers and related enzymes in vertebrate rod outer segments

Giusto

Pasquaré

Salvador

et al. 2010

Journal of Lipid Research

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outer segment of the cell is comprised of a stack of many hundreds of densely packed discs, each of which represents a double layer of infolded plasma membrane. It has been suggested that phospholipid moieties in these membranes are metabolically active and are closely involved in the generation of physiological mediators. Modifi cations in the metabolism of photoreceptor membrane glycerolipids have been linked to the transduction of visual stimuli ( 3 ). Rod outer segment (ROS) membranes are composed of 50% protein and 50% lipid, by weight ( 2 ). Phospholipids and cholesterol represent nearly 90-95% and 4-6% (w/w) of total lipids, respectively. Rhodopsin accounts for at least 80-85% (w/w) of the total proteins ( 4-6 ) in disc ROS membranes. The rest are mainly proteins involved in phototransduction. The composition of mammalian ROS membrane phospholipids indicates that the major phospholipids are phosphatidylethanolamine (PE) and phosphatidylcholine (PC), along with relatively large amounts of phosphatidylserine (PS) (15%). Small amounts (less than 1% each) of sphingomyelin, phosphatidylinositol (PI) and phosphatidic acid (PA) are found in these membranes ( 7-13 ). In vertebrates, cholesterol (exclusively as the free sterol) is the principal neutral lipid, comprising The vertebrate photoreceptor cell is composed of different cellular compartments [outer segment (OS), inner segment (IS), and synaptic terminal (ST)], all of which differ in their lipid and protein content ( 1, 2 ). The photoreceptor is a highly specialized cell that responds to light stimulus and transmits this response to adjoining neurons for ultimate relay to the visual centers of the brain. TheThis work was supported

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“…For example, some isoforms (e.g. ␣, ␥, , and ) of mammalian DAG kinase are phosphorylated by protein kinase C (91)(92)(93)(94) or by the tyrosine kinase Src (95) to stimulate their activity and function in lipid signaling. Although CKII is evolutionarily conserved (54), it is unclear whether the protein kinase phosphorylates mammalian DAG kinase enzymes.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae

Qiu

Hassaninasab

Han

et al. 2016

Journal of Biological Chemistry

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Edited by Dennis VoelkerIn the yeast Saccharomyces cerevisiae, Dgk1 diacylglycerol (DAG) kinase catalyzes the CTP-dependent phosphorylation of DAG to form phosphatidic acid (PA). The enzyme in conjunction with Pah1 PA phosphatase controls the levels of PA and DAG for the synthesis of triacylglycerol and membrane phospholipids, the growth of the nuclear/endoplasmic reticulum membrane, and the formation of lipid droplets. Little is known about how DAG kinase activity is regulated by posttranslational modification. In this work, we examined the phosphorylation of Dgk1 DAG kinase by casein kinase II (CKII). When phosphate groups were globally reduced using nonspecific alkaline phosphatase, Triton X-100-solubilized membranes from DGK1-overexpressing cells showed a 7.7-fold reduction in DAG kinase activity; the reduced enzyme activity could be increased 5.5-fold by treatment with CKII. Dgk1(1-77) expressed heterologously in Escherichia coli was phosphorylated by CKII on a serine residue, and its phosphorylation was dependent on time as well as on the concentrations of CKII, ATP, and Dgk1(1-77). We used site-specific mutagenesis, coupled with phosphorylation analysis and phosphopeptide mapping, to identify Ser-45 and Ser-46 of Dgk1 as the CKII target sites, with Ser-46 being the major phosphorylation site. In vivo, the S46A and S45A/S46A mutations of Dgk1 abolished the stationary phase-dependent stimulation of DAG kinase activity. In addition, the phosphorylation-deficient mutations decreased Dgk1 function in PA production and in eliciting pah1⌬ phenotypes, such as the expansion of the nuclear/endoplasmic reticulum membrane, reduced lipid droplet formation, and temperaturesensitivity.ThisworkdemonstratesthattheCKII-mediatedphosphorylation of Dgk1 regulates its function in the production of PA.The yeast 2 Dgk1 DAG 3 kinase, an integral membrane enzyme catalyzing the CTP-dependent phosphorylation of DAG to PA (1, 2), and Pah1 PA phosphatase, a peripheral membrane enzyme catalyzing the Mg 2ϩ -dependent dephosphorylation of PA to DAG (3), have emerged as key regulators of the essential lipid intermediates PA and DAG ( Fig. 1) (4, 5). PA is used for the synthesis of all membrane phospholipids via CDP-DAG (CDP-DAG Pathway) or DAG (Kennedy Pathway) and for the synthesis of TAG via DAG (4, 5) (Fig. 1). In addition, the mobilization of TAG to produce DAG and its subsequent phosphorylation to PA for the synthesis of membrane phospholipids (Fig. 1) play an important role in the growth resumption of yeast cells that exit stasis (e.g. stationary phase) (6). Moreover, PA and DAG serve as signaling molecules that control transcription, membrane proliferation, vesicular trafficking, and the activation of cell growth (7-16).Disturbing the PA/DAG balance in yeast, as caused by the lack of Pah1 PA phosphatase activity, results in the abnormal regulation of phospholipid synthesis gene expression and phospholipid content, the aberrant growth of the nuclear/ER membrane, vacuole fragmentation, a defect in lipid droplet formation, an acute se...

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Phosphorylation and Up-regulation of Diacylglycerol Kinase γ via Its Interaction with Protein Kinase Cγ

Cited by 39 publications

References 45 publications

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 kDa (PRAS40) Is a Physiological Substrate of Mammalian Target of Rapamycin Complex 1

Lipid second messengers and related enzymes in vertebrate rod outer segments

Phosphorylation of Dgk1 Diacylglycerol Kinase by Casein Kinase II Regulates Phosphatidic Acid Production in Saccharomyces cerevisiae

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