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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

LPAAT-β siRNA treatment results in enhanced nuclear localization of Lipin 1.Immunofluoresence staining (A) for Lipin 1 (red) with the nuclei counterstained with DAPI (blue) and overlay (merge) of MiaPaCa2 cells treated with: DMSO and 500 nM Torin-1 for 24 hours; control siRNA (nt), 10 nM LP-1, 25 nM LP-2 and 10 nM LP-4 for 48 hr. B) Quantitation of nuclear Lipin 1 fluoresence intensity from (A). Statistical significance was calculated using Student’s t-test: DMSO (*) vs Torin-1, p < 0.0001; siRNA nt (**) vs LP-1, p = 0.0005; vs LP-2, p = 0.05; vs. LP-4, p = 0.02. Results are representative of three independent experiments.
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pone-0078632-g007: LPAAT-β siRNA treatment results in enhanced nuclear localization of Lipin 1.Immunofluoresence staining (A) for Lipin 1 (red) with the nuclei counterstained with DAPI (blue) and overlay (merge) of MiaPaCa2 cells treated with: DMSO and 500 nM Torin-1 for 24 hours; control siRNA (nt), 10 nM LP-1, 25 nM LP-2 and 10 nM LP-4 for 48 hr. B) Quantitation of nuclear Lipin 1 fluoresence intensity from (A). Statistical significance was calculated using Student’s t-test: DMSO (*) vs Torin-1, p < 0.0001; siRNA nt (**) vs LP-1, p = 0.0005; vs LP-2, p = 0.05; vs. LP-4, p = 0.02. Results are representative of three independent experiments.

Mentions: We then transfected MiaPaCa2 cells for 72 hours with either non-targeting siRNA or with siRNA to LPAAT-β (LP-1 and LP-4). Figure 6B reveals that MiaPaCa2 cells treated with LP-4 showed statistically significant nuclear elongation (p < 0.0001), as determined by both measurements used. Likewise, MiaPaCa2 cells transfected for 48 hours with LP-1 or 72 hours with LP-2 also showed statistically significant increases in these measures of nuclear eccentricity (Figure S4). We next assessed the nuclear localization of Lipin 1. Immunostaining of MiaPaCa2 cells treated for 24 hours with Torin-1 show an enhanced localization of Lipin 1 in the nuclei of the cells compared to a diffuse staining of Lipin 1 in DMSO treated cells (Figure 7). Similarly, cells treated for 48 hours with siRNA to LPAAT-β also have enhanced nuclear Lipin 1 staining (Figure 7) that is statistically significant and comparable to the effect of Torin-1. Taken together, these data provide further support for the hypothesis that PA from LPAAT-β regulates the mTOR signaling nexus.


Lysophosphatidic acid acyltransferase beta regulates mTOR signaling.

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

LPAAT-β siRNA treatment results in enhanced nuclear localization of Lipin 1.Immunofluoresence staining (A) for Lipin 1 (red) with the nuclei counterstained with DAPI (blue) and overlay (merge) of MiaPaCa2 cells treated with: DMSO and 500 nM Torin-1 for 24 hours; control siRNA (nt), 10 nM LP-1, 25 nM LP-2 and 10 nM LP-4 for 48 hr. B) Quantitation of nuclear Lipin 1 fluoresence intensity from (A). Statistical significance was calculated using Student’s t-test: DMSO (*) vs Torin-1, p < 0.0001; siRNA nt (**) vs LP-1, p = 0.0005; vs LP-2, p = 0.05; vs. LP-4, p = 0.02. Results are representative of three independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3814986&req=5

pone-0078632-g007: LPAAT-β siRNA treatment results in enhanced nuclear localization of Lipin 1.Immunofluoresence staining (A) for Lipin 1 (red) with the nuclei counterstained with DAPI (blue) and overlay (merge) of MiaPaCa2 cells treated with: DMSO and 500 nM Torin-1 for 24 hours; control siRNA (nt), 10 nM LP-1, 25 nM LP-2 and 10 nM LP-4 for 48 hr. B) Quantitation of nuclear Lipin 1 fluoresence intensity from (A). Statistical significance was calculated using Student’s t-test: DMSO (*) vs Torin-1, p < 0.0001; siRNA nt (**) vs LP-1, p = 0.0005; vs LP-2, p = 0.05; vs. LP-4, p = 0.02. Results are representative of three independent experiments.
Mentions: We then transfected MiaPaCa2 cells for 72 hours with either non-targeting siRNA or with siRNA to LPAAT-β (LP-1 and LP-4). Figure 6B reveals that MiaPaCa2 cells treated with LP-4 showed statistically significant nuclear elongation (p < 0.0001), as determined by both measurements used. Likewise, MiaPaCa2 cells transfected for 48 hours with LP-1 or 72 hours with LP-2 also showed statistically significant increases in these measures of nuclear eccentricity (Figure S4). We next assessed the nuclear localization of Lipin 1. Immunostaining of MiaPaCa2 cells treated for 24 hours with Torin-1 show an enhanced localization of Lipin 1 in the nuclei of the cells compared to a diffuse staining of Lipin 1 in DMSO treated cells (Figure 7). Similarly, cells treated for 48 hours with siRNA to LPAAT-β also have enhanced nuclear Lipin 1 staining (Figure 7) that is statistically significant and comparable to the effect of Torin-1. Taken together, these data provide further support for the hypothesis that PA from LPAAT-β regulates the mTOR signaling nexus.

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