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Lysophosphatidic acid acyltransferase beta regulates mTOR signaling.

Blaskovich MA, Yendluri V, Lawrence HR, Lawrence NJ, Sebti SM, Springett GM - PLoS ONE (2013)

Bottom Line: Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down.Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function.Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Therapeutics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America.

ABSTRACT
Lysophosphatidic acid acyltransferase (LPAAT-β) is a phosphatidic acid (PA) generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.

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Related in: MedlinePlus

Treatment with LPAAT-β siRNA results in inhibition of phosphorylation of mTOR effector proteins 4E-BP1, S6K, and AKT.AsPC-1 (A) and MiaPaCa2 (B) cells were treated for 72 hr with 25 nM LP-1, LP-4, non-targeting siRNA (nt), or a mock transfectant (m). Western blots were done with the indicated antibodies to mTOR effector proteins and mTOR-specific phosphorylation sites, as well as a Vinculin loading control. The graphs show densitometric quantitation of the phosphorylated bands normalized to both Vinculin and their corresponding whole protein. Results are representative of three independent experiments.
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pone-0078632-g004: Treatment with LPAAT-β siRNA results in inhibition of phosphorylation of mTOR effector proteins 4E-BP1, S6K, and AKT.AsPC-1 (A) and MiaPaCa2 (B) cells were treated for 72 hr with 25 nM LP-1, LP-4, non-targeting siRNA (nt), or a mock transfectant (m). Western blots were done with the indicated antibodies to mTOR effector proteins and mTOR-specific phosphorylation sites, as well as a Vinculin loading control. The graphs show densitometric quantitation of the phosphorylated bands normalized to both Vinculin and their corresponding whole protein. Results are representative of three independent experiments.

Mentions: We transfected AsPC-1 and MiaPaCa2 cells with LPAAT-β or non-targeting siRNA and then probed for the mTOR kinase phosphorylation sites of S6K, 4E-BP1, and AKT by Western blotting (Figure 4A). LP-1 and LP-4 inhibited Ser65 phosphorylation of 4E-BP1 by 80% and 50%, respectively in AsPC-1 compared to non-targeting control siRNA (Figure 4A, 4D). In MiaPaCa2 cells the inhibition of phospho-4E-BP1 was 70% and 40% respectively (Figure 4A, 4E). The other mTORC1 kinase target, S6K, was also inhibited at Thr389 by 75% and 50% (Figure 4A, 4D) in AsPC-1 and 80% and 40% in MiaPaCa2 (Figure 4A, 4E). Phosphorylation of the mTORC2 kinase substrate, AKT was inhibited at Ser473 by 50% and 30% respectively in AsPC-1 (Figure 4A, 4B), while in MiaPaCa2 the degree of inhibition was 60% and 40%, respectively (Figure 4A, 4E). The degree of inhibition of signaling correlated well with the extent of knockdown LPAAT- β expression in both cell lines (Figure 4B, 4C). These results suggest that LPAAT-β directly regulates the major mTOR signaling targets in pancreatic cancer cells.


Lysophosphatidic acid acyltransferase beta regulates mTOR signaling.

Blaskovich MA, Yendluri V, Lawrence HR, Lawrence NJ, Sebti SM, Springett GM - PLoS ONE (2013)

Treatment with LPAAT-β siRNA results in inhibition of phosphorylation of mTOR effector proteins 4E-BP1, S6K, and AKT.AsPC-1 (A) and MiaPaCa2 (B) cells were treated for 72 hr with 25 nM LP-1, LP-4, non-targeting siRNA (nt), or a mock transfectant (m). Western blots were done with the indicated antibodies to mTOR effector proteins and mTOR-specific phosphorylation sites, as well as a Vinculin loading control. The graphs show densitometric quantitation of the phosphorylated bands normalized to both Vinculin and their corresponding whole protein. Results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3814986&req=5

pone-0078632-g004: Treatment with LPAAT-β siRNA results in inhibition of phosphorylation of mTOR effector proteins 4E-BP1, S6K, and AKT.AsPC-1 (A) and MiaPaCa2 (B) cells were treated for 72 hr with 25 nM LP-1, LP-4, non-targeting siRNA (nt), or a mock transfectant (m). Western blots were done with the indicated antibodies to mTOR effector proteins and mTOR-specific phosphorylation sites, as well as a Vinculin loading control. The graphs show densitometric quantitation of the phosphorylated bands normalized to both Vinculin and their corresponding whole protein. Results are representative of three independent experiments.
Mentions: We transfected AsPC-1 and MiaPaCa2 cells with LPAAT-β or non-targeting siRNA and then probed for the mTOR kinase phosphorylation sites of S6K, 4E-BP1, and AKT by Western blotting (Figure 4A). LP-1 and LP-4 inhibited Ser65 phosphorylation of 4E-BP1 by 80% and 50%, respectively in AsPC-1 compared to non-targeting control siRNA (Figure 4A, 4D). In MiaPaCa2 cells the inhibition of phospho-4E-BP1 was 70% and 40% respectively (Figure 4A, 4E). The other mTORC1 kinase target, S6K, was also inhibited at Thr389 by 75% and 50% (Figure 4A, 4D) in AsPC-1 and 80% and 40% in MiaPaCa2 (Figure 4A, 4E). Phosphorylation of the mTORC2 kinase substrate, AKT was inhibited at Ser473 by 50% and 30% respectively in AsPC-1 (Figure 4A, 4B), while in MiaPaCa2 the degree of inhibition was 60% and 40%, respectively (Figure 4A, 4E). The degree of inhibition of signaling correlated well with the extent of knockdown LPAAT- β expression in both cell lines (Figure 4B, 4C). These results suggest that LPAAT-β directly regulates the major mTOR signaling targets in pancreatic cancer cells.

Bottom Line: Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down.Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function.Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Therapeutics, H. Lee Moffitt Cancer Center and Research Institute, Tampa, Florida, United States of America.

ABSTRACT
Lysophosphatidic acid acyltransferase (LPAAT-β) is a phosphatidic acid (PA) generating enzyme that plays an essential role in triglyceride synthesis. However, LPAAT-β is now being studied as an important regulator of cell growth and differentiation and as a potential therapeutic target in cancer since PA is necessary for the activity of key proteins such as Raf, PKC-ζ and mTOR. In this report we determine the effect of LPAAT-β silencing with siRNA in pancreatic adenocarcinoma cell lines. We show for the first time that LPAAT-β knockdown inhibits proliferation and anchorage-independent growth of pancreatic cancer cells. This is associated with inhibition of signaling by mTOR as determined by levels of mTORC1- and mTORC2-specific phosphorylation sites on 4E-BP1, S6K and Akt. Since PA regulates the activity of mTOR by modulating its binding to FKBP38, we explored the possibility that LPAAT-β might regulate mTOR by affecting its association with FKBP38. Coimmunoprecipitation studies of FKBP38 with mTOR show increased levels of FKBP38 associated with mTOR when LPAAT-β protein levels are knocked down. Furthermore, depletion of LPAAT-β results in increased Lipin 1 nuclear localization which is associated with increased nuclear eccentricity, a nuclear shape change that is dependent on mTOR, further confirming the ability of LPAAT-β to regulate mTOR function. Our results provide support for the hypothesis that PA generated by LPAAT-β regulates mTOR signaling. We discuss the implications of these findings for using LPAAT-β as a therapeutic target.

Show MeSH
Related in: MedlinePlus