Lipin 2 Binds Phosphatidic Acid by the Electrostatic Hydrogen Bond Switch Mechanism Independent of Phosphorylation

Eaton, James M.; Takkellapati, Sankeerth; Lawrence, Robert; McQueeney, Kelley E.; Boroda, Salome; Mullins, Garrett R.; Sherwood, Samantha G.; Finck, Brian N.; Villén, Judit; Harris, Thurl E.

doi:10.1074/jbc.m114.547604

Cited by 30 publications

(56 citation statements)

References 41 publications

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“…However, a sampling of k cat /K m values from this analysis ( (20,(22)(23)(24), similar to those of C1 and C2 enzymes (21,22,(25)(26)(27). Overall, the finding that C0 members, which have the most minimal cap inserts, are equally efficient and more specific than the other subfamilies suggests that incorporation of additional domains leads to the expansion of the substrate repertoire in the HADSF.…”

Section: Resultsmentioning

confidence: 93%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

136

139

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Large-scale activity profiling of enzyme superfamilies provides information about cellular functions as well as the intrinsic binding capabilities of conserved folds. Herein, the functional space of the ubiquitous haloalkanoate dehalogenase superfamily (HADSF) was revealed by screening a customized substrate library against >200 enzymes from representative prokaryotic species, enabling inferred annotation of ∼35% of the HADSF. An extremely high level of substrate ambiguity was revealed, with the majority of HADSF enzymes using more than five substrates. Substrate profiling allowed assignment of function to previously unannotated enzymes with known structure, uncovered potential new pathways, and identified isofunctional orthologs from evolutionarily distant taxonomic groups. Intriguingly, the HADSF subfamily having the least structural elaboration of the Rossmann fold catalytic domain was the most specific, consistent with the concept that domain insertions drive the evolution of new functions and that the broad specificity observed in HADSF may be a relic of this process.evolution | specificity | phosphatase | substrate screen | promiscuity S ince the first genomes were sequenced, there has been an exponential increase in the number of protein sequences deposited into databases worldwide. At the time of this writing the UniProtKB/TrEMBL database contains over 32 million protein sequences. Although this increase in sequence data has dramatically enhanced our understanding of the genomic organization of organisms, as the number of protein sequences grows, the proportion of firm functional assignments diminishes. Traditionally, methods of functional annotation involve comparing sequence identity between experimentally characterized proteins and newly sequenced ones, typically via BLAST (1). In cases where significant sequence similarity cannot be ascertained, proteins are annotated as "hypothetical" or "putative." Moreover, the decrease in sequence identity leads to an increased uncertainty in functional assignment, especially as the phylogenetic distance between organisms grows, limiting iso-functional ortholog discovery.As the number of newly sequenced genomes grows larger, more protein sequences are likely to be misannotated, oftentimes resulting in the propagation of incorrect functional annotation across newly identified sequences. To tackle the problem of unannotated or misannotated proteins, newer methods for computational assignment have been created with varying degrees of success (2). Although these methods outperform historical methods, continued improvement is necessary to ensure accurate annotation of function (2). A greater swath of functional space can be covered by screening substrates in a high-throughput manner on multiple enzymes from a family (3, 4). Family-wide substrate profiling offers a data-rich resource. The use of sparse screening of sequence space and a diversified library permits the determination of substrate specificity profiles to provide a familywide view of the range of substrates...

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Section: Resultsmentioning

confidence: 93%

Panoramic view of a superfamily of phosphatases through substrate profiling

Huang

Pandya

Liu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

136

139

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“…Although the intrinsic catalytic activity of lipin 2 is lower than lipin 1 ( 4 ), lipin 2 is subject to less regulation by phosphorylation ( 50 ). Lipin 2 protein abundance was increased in Alb-Lpin1 Ϫ / Ϫ liver.…”

Section: Discussionmentioning

confidence: 92%

Liver-specific loss of lipin-1-mediated phosphatidic acid phosphatase activity does not mitigate intrahepatic TG accumulation in mice

Schweitzer

Chen

Gan

et al. 2015

Journal of Lipid Research

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This article is available online at http://www.jlr.org states and may be linked to other abnormalities in liver metabolism and function. Indeed, in a subset of individuals, hepatic steatosis can progress to liver injury, dysfunction, and failure. Intrahepatic lipid accumulation is also usually highly correlated with systemic insulin resistance, hyperglycemia, dyslipidemias, and risk of cardiovascular disease. Hepatic steatosis is extremely prevalent in obese individuals, and with the epidemic of obesity, the occurrence of nonalcoholic fatty liver disease has risen dramatically, becoming the most common cause of liver disease in the United States ( 1, 2 ).The primary storage form of lipid is TG, which, in the liver, is predominantly synthesized via the sequential acylation and dephosphorylation of glycerol-3-phosphate. In higher organisms, three genes ( Lpin1 , Lpin2 , and Lpin3 ) encode canonical enzymes that catalyze the Mg 2+ -dependent dephosphorylation of phosphatidic acid (PA) to form diacylglycerol (DAG) at the endoplasmic reticulum ( 3, 4 ). The phosphatidic acid phosphohydrolase (PAP) reaction is not only the penultimate step in TG synthesis, but also a key metabolic branch point. Alterations in PA and DAG concentrations have been linked to regulation of important intracellular signaling cascades including protein kinase C ( 5, 6 ), protein kinase A ( 7 ), ERK MAPK kinase ( 8 ), and the molecular target of rapamycin ( 9-11 ). The regulation of PA and DAG concentrations has potentially important implications for hepatic nutrient homeostasis under conditions of fasting and overnutrition. Indeed, acute RNA interference (RNAi)-mediated depletion of lipin 1 or lipin 2 in mice fed a high-fat diet attenuated hepatic steatosis and led to improvements in hepatic insulin sensitivity ( 12, 13 ).Lipins are not integral membrane proteins, and lipin 1 can translocate into the nucleus and also interact with Abstract Lipin proteins (lipin 1, 2, and 3) regulate glycerolipid homeostasis by acting as phosphatidic acid phosphohydrolase (PAP) enzymes in the TG synthesis pathway and by regulating DNA-bound transcription factors to control gene transcription. Hepatic PAP activity could contribute to hepatic fat accumulation in response to physiological and pathophysiological stimuli. To examine the role of lipin 1 in regulating hepatic lipid metabolism, we generated mice that are defi cient in lipin-1-encoded PAP activity in a liver-specifi c manner (Alb-Lpin1 ؊ / ؊ mice). This allele of lipin 1 was still able to transcriptionally regulate the expression of its target genes encoding fatty acid oxidation enzymes, and the expression of these genes was not affected in Alb-Lpin1 ؊ / ؊ mouse liver. Hepatic PAP activity was signifi cantly reduced in mice with liver-specifi c lipin 1 defi ciency. However, hepatocytes from Alb-Lpin1 ؊ / ؊ mice had normal rates of TG synthesis, and steady-state hepatic TG levels were unaffected under fed and fasted conditions. Furthermore, Alb-Lpin1 ؊ / ؊ mice were not protected from intrahepatic accum...

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“…A wealth of data now supports our understanding of lipin phosphorylation and its ability to regulate localization and activity (5,66,87,(94)(95)(96)(97). Other lipin kinases remain to be identified and the direct effect of phosphorylation on enzymatic activity needs to be further elucidated (95).…”

Section: Lipins -Biochemical Regulation I Insulin and Phosphorylationmentioning

confidence: 90%

“…More specifically, inhibition of mTOR and reduction in the phosphorylation of lipin 1, allow its translocation to the ER membrane and nucleus (66,85,93,96,97). We have shown that while lipin 1 is regulated by phosphorylation in vitro, the activity and localization of lipin 2 are not (94).…”

Section: Introductionmentioning

confidence: 87%

“…Other lipin kinases remain to be identified and the direct effect of phosphorylation on enzymatic activity needs to be further elucidated (95). The current consensus is that phosphorylation downstream of insulin signaling negatively regulates the activity and cellular localization of lipin 1, but not lipin 2, by sequestering some of the protein in the cytosol (66,94,96,97).…”

Section: Lipins -Biochemical Regulation I Insulin and Phosphorylationmentioning

confidence: 99%

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Repositioning of Ritanserin for the Inhibition of DGK-Alpha and the Role of the Polybasic Domain in the Phosphoregulation of Lipins 1 and 3

Boroda

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II ABSTRACTThe diversity and multi-functionality of cellular lipids is astounding. At the hub of phospho-and neutral lipid flux are two lipids with many effector proteins: diacylglycerol (DAG) and phosphatidic acid (PA). Enzymes which can simultaneously regulate both of their levels are diacylglycerol kinases (DGKs) and lipins. The former phosphorylates DAG to yield PA and the latter dephosphorylates PA to generate DAG. The work herein approaches the study of DGKs and lipins from two different perspectives.First, we have built upon previous work, which established DGKα as a therapeutic target in glioblastoma multiforme (GBM), and have repositioned ritanserin, a serotonin receptor (5-HTR) antagonist, as a pharmacological tool for attenuating DGK activity. We have also elucidated the polypharmacology of other DGK inhibitors, and shown that they are potent 5-HTR antagonists.Second, we hypothesized that the varying PA-binding domains, or polybasic domains (PBDs), of each lipin are involved in the phosphoregulatory differences among these enzymes. We show, for the first time, that insertion of the lipin 3 PBD in the lipin 1 enzyme eliminates the phosphoregulation of lipin 1. Conversely, the insertion of lipin 1 PBD into the lipin 3 protein allows phosphorylated residues on lipin 3 to negatively regulate its activity in vitro. These data suggest that the differences in the amino acid sequence between PBDs of these two proteins are sufficient to determine whether or not the enzymes can be controlled by intramolecular phosphoregulation III ACKNOWLEDGEMENTS

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Lipin 2 Binds Phosphatidic Acid by the Electrostatic Hydrogen Bond Switch Mechanism Independent of Phosphorylation

Cited by 30 publications

References 41 publications

Panoramic view of a superfamily of phosphatases through substrate profiling

Panoramic view of a superfamily of phosphatases through substrate profiling

Liver-specific loss of lipin-1-mediated phosphatidic acid phosphatase activity does not mitigate intrahepatic TG accumulation in mice

Repositioning of Ritanserin for the Inhibition of DGK-Alpha and the Role of the Polybasic Domain in the Phosphoregulation of Lipins 1 and 3

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