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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 increased nuclear eccentricity.MiaPaCa2 cells were treated (A) for 24 hr with 500 nM Torin-1 (* p < 0.0001), 10 nM rapamycin, or DMSO vehicle and (B) for 72 hr with 25 nM LP-4 (* p = 0.05) or non-targeting (nt) siRNA. Nuclei were stained with antibody to Lamin A to highlight the nuclear membrane as well as counterstained with DAPI. Analysis of the images using Definiens Tissue Studio software generated the length and width of individual nuclei, which were then analyzed for enhanced nuclear eccentricity as described in Materials and Methods. Graphs show the frequency distribution of nuclear eccentricity and nuclear l/w normalized to cell number. Statistical significance was determined using Student’s t-test. Results are representative of four independent experiments.
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pone-0078632-g006: LPAAT-β siRNA treatment results in increased nuclear eccentricity.MiaPaCa2 cells were treated (A) for 24 hr with 500 nM Torin-1 (* p < 0.0001), 10 nM rapamycin, or DMSO vehicle and (B) for 72 hr with 25 nM LP-4 (* p = 0.05) or non-targeting (nt) siRNA. Nuclei were stained with antibody to Lamin A to highlight the nuclear membrane as well as counterstained with DAPI. Analysis of the images using Definiens Tissue Studio software generated the length and width of individual nuclei, which were then analyzed for enhanced nuclear eccentricity as described in Materials and Methods. Graphs show the frequency distribution of nuclear eccentricity and nuclear l/w normalized to cell number. Statistical significance was determined using Student’s t-test. Results are representative of four independent experiments.

Mentions: To test this, we first established that nuclear eccentricity in MiaPaCa2 cells was increased upon Torin-1 treatment but not rapamycin, as shown by Peterson et al. in NIH 3T3 cells overexpressing Lipin 1[30]. Cells plated onto coverslips were treated for 24 hours either with vehicle control, Torin-1, or rapamycin. Figure 6A shows that MiaPaCa2 cells treated with Torin-1 show the characteristic increase in nuclear eccentricity described previously. To quantify this morphological change, we used two measures of nuclear shape: the formula for eccentricity utilized by Sabatini and colleagues [30], eccentricity = 1-2/((a/p)+1) [30] (see Materials and Methods for an explanation of this formula); and the simple ratio of nuclear length/width. Treatment of MiaPaCa2 cells with Torin-1 shows a measureable, statistically significant increase in nuclear eccentricity as calculated by both measures (p < 0.05 as determined by Student’s t-test).


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 increased nuclear eccentricity.MiaPaCa2 cells were treated (A) for 24 hr with 500 nM Torin-1 (* p < 0.0001), 10 nM rapamycin, or DMSO vehicle and (B) for 72 hr with 25 nM LP-4 (* p = 0.05) or non-targeting (nt) siRNA. Nuclei were stained with antibody to Lamin A to highlight the nuclear membrane as well as counterstained with DAPI. Analysis of the images using Definiens Tissue Studio software generated the length and width of individual nuclei, which were then analyzed for enhanced nuclear eccentricity as described in Materials and Methods. Graphs show the frequency distribution of nuclear eccentricity and nuclear l/w normalized to cell number. Statistical significance was determined using Student’s t-test. Results are representative of four independent experiments.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0078632-g006: LPAAT-β siRNA treatment results in increased nuclear eccentricity.MiaPaCa2 cells were treated (A) for 24 hr with 500 nM Torin-1 (* p < 0.0001), 10 nM rapamycin, or DMSO vehicle and (B) for 72 hr with 25 nM LP-4 (* p = 0.05) or non-targeting (nt) siRNA. Nuclei were stained with antibody to Lamin A to highlight the nuclear membrane as well as counterstained with DAPI. Analysis of the images using Definiens Tissue Studio software generated the length and width of individual nuclei, which were then analyzed for enhanced nuclear eccentricity as described in Materials and Methods. Graphs show the frequency distribution of nuclear eccentricity and nuclear l/w normalized to cell number. Statistical significance was determined using Student’s t-test. Results are representative of four independent experiments.
Mentions: To test this, we first established that nuclear eccentricity in MiaPaCa2 cells was increased upon Torin-1 treatment but not rapamycin, as shown by Peterson et al. in NIH 3T3 cells overexpressing Lipin 1[30]. Cells plated onto coverslips were treated for 24 hours either with vehicle control, Torin-1, or rapamycin. Figure 6A shows that MiaPaCa2 cells treated with Torin-1 show the characteristic increase in nuclear eccentricity described previously. To quantify this morphological change, we used two measures of nuclear shape: the formula for eccentricity utilized by Sabatini and colleagues [30], eccentricity = 1-2/((a/p)+1) [30] (see Materials and Methods for an explanation of this formula); and the simple ratio of nuclear length/width. Treatment of MiaPaCa2 cells with Torin-1 shows a measureable, statistically significant increase in nuclear eccentricity as calculated by both measures (p < 0.05 as determined by Student’s t-test).

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