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Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway.

Wang CH, Shyu RY, Wu CC, Tsai TC, Wang LK, Chen ML, Jiang SY, Tsai FM - J. Biomed. Sci. (2014)

Bottom Line: AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor.Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation.Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Research, Taipei Tzuchi Hospital, The Buddhist Tzuchi Medical Foundation, New Taipei City, Taiwan. afu2215@gmail.com.

ABSTRACT

Background: H-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family. Previous studies showed that H-rev107 exhibits phospholipase A/acyltransferase (PLA/AT) activity and downregulates H-RAS expression. However, the mode of action and the site of inhibition of H-RAS by H-rev107 are still unknown.

Results: Our results indicate that H-rev107 was co-precipitated with H-RAS and downregulated the levels of activated RAS (RAS-GTP) and ELK1-mediated transactivation in epidermal growth factor-stimulated and H-RAS-cotransfected HtTA cervical cancer cells. Furthermore, an acyl-biotin exchange assay demonstrated that H-rev107 reduced H-RAS palmitoylation. H-rev107 has been shown to be a PLA/AT that is involved in phospholipid metabolism. Treating cells with the PLA/AT inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) or methyl arachidonyl fluorophosphate (MAFP) alleviated H-rev107-induced downregulation of the levels of acylated H-RAS. AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor. In contrast, treating cells with the acyl-protein thioesterase inhibitor palmostatin B enhanced H-rev107-mediated downregulation of acylated H-RAS in H-rev107-expressing cells. Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation. These results suggest that H-rev107 decreases H-RAS activity through its PLA/AT activity to modulate H-RAS acylation.

Conclusions: We made the novel observation that H-rev107 decrease in the steady state levels of H-RAS palmitoylation through the phospholipase A/acyltransferase activity. H-rev107 is likely to suppress activation of the RAS signaling pathway by reducing the levels of palmitoylated H-RAS, which decreases the levels of GTP-bound H-RAS and also the activation of downstream molecules. Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.

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H-rev107 was associated with H-RAS and inhibited its palmitoylation. (A) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with H-rev107-myc or the control vector. Cell lysates were prepared as described in Methods. The interaction between H-rev107 and RAS was analyzed by immunoprecipitation followed by Western blot analysis. (B) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with 3 μg of H-rev107-myc or the control vector. Cell lysates were prepared, and acyl-biotin exchange analysis of H-RAS was performed as described in Methods. Aliquots containing 5 μg of protein including acylated RAS were biotinylated and then processed with streptavidin agarose resin followed by Western blot analysis. The input consisted of 300 ng of protein from the acyl-biotin exchange that was loaded. HC: heavy chain; NH2OH: hydroxylamine.
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Figure 2: H-rev107 was associated with H-RAS and inhibited its palmitoylation. (A) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with H-rev107-myc or the control vector. Cell lysates were prepared as described in Methods. The interaction between H-rev107 and RAS was analyzed by immunoprecipitation followed by Western blot analysis. (B) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with 3 μg of H-rev107-myc or the control vector. Cell lysates were prepared, and acyl-biotin exchange analysis of H-RAS was performed as described in Methods. Aliquots containing 5 μg of protein including acylated RAS were biotinylated and then processed with streptavidin agarose resin followed by Western blot analysis. The input consisted of 300 ng of protein from the acyl-biotin exchange that was loaded. HC: heavy chain; NH2OH: hydroxylamine.

Mentions: The results above suggest that H-rev107 can inhibit H-RAS activation. We then analyzed whether H-RAS is the acylated target of H-rev107. We first performed co-immunoprecipitation with lysates of HtTA cells that coexpressed H-RAS along with empty vector or H-rev107-myc fusion protein. Our analysis revealed that H-RAS was immunoprecipitated along with H-rev107 fusion protein using anti-MYC antibody against the MYC epitope of the H-rev107 fusion protein. Similarly, H-rev107 was detected in the H-RAS immunoprecipitate (Figure 2A).


Phospholipase A/Acyltransferase enzyme activity of H-rev107 inhibits the H-RAS signaling pathway.

Wang CH, Shyu RY, Wu CC, Tsai TC, Wang LK, Chen ML, Jiang SY, Tsai FM - J. Biomed. Sci. (2014)

H-rev107 was associated with H-RAS and inhibited its palmitoylation. (A) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with H-rev107-myc or the control vector. Cell lysates were prepared as described in Methods. The interaction between H-rev107 and RAS was analyzed by immunoprecipitation followed by Western blot analysis. (B) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with 3 μg of H-rev107-myc or the control vector. Cell lysates were prepared, and acyl-biotin exchange analysis of H-RAS was performed as described in Methods. Aliquots containing 5 μg of protein including acylated RAS were biotinylated and then processed with streptavidin agarose resin followed by Western blot analysis. The input consisted of 300 ng of protein from the acyl-biotin exchange that was loaded. HC: heavy chain; NH2OH: hydroxylamine.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4012743&req=5

Figure 2: H-rev107 was associated with H-RAS and inhibited its palmitoylation. (A) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with H-rev107-myc or the control vector. Cell lysates were prepared as described in Methods. The interaction between H-rev107 and RAS was analyzed by immunoprecipitation followed by Western blot analysis. (B) HtTA cells plated in a 10-cm dish were transfected for 24 h with 0.1 μg of H-RAS along with 3 μg of H-rev107-myc or the control vector. Cell lysates were prepared, and acyl-biotin exchange analysis of H-RAS was performed as described in Methods. Aliquots containing 5 μg of protein including acylated RAS were biotinylated and then processed with streptavidin agarose resin followed by Western blot analysis. The input consisted of 300 ng of protein from the acyl-biotin exchange that was loaded. HC: heavy chain; NH2OH: hydroxylamine.
Mentions: The results above suggest that H-rev107 can inhibit H-RAS activation. We then analyzed whether H-RAS is the acylated target of H-rev107. We first performed co-immunoprecipitation with lysates of HtTA cells that coexpressed H-RAS along with empty vector or H-rev107-myc fusion protein. Our analysis revealed that H-RAS was immunoprecipitated along with H-rev107 fusion protein using anti-MYC antibody against the MYC epitope of the H-rev107 fusion protein. Similarly, H-rev107 was detected in the H-RAS immunoprecipitate (Figure 2A).

Bottom Line: AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor.Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation.Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Research, Taipei Tzuchi Hospital, The Buddhist Tzuchi Medical Foundation, New Taipei City, Taiwan. afu2215@gmail.com.

ABSTRACT

Background: H-rev107, also called HRASLS3 or PLA2G16, is a member of the HREV107 type II tumor suppressor gene family. Previous studies showed that H-rev107 exhibits phospholipase A/acyltransferase (PLA/AT) activity and downregulates H-RAS expression. However, the mode of action and the site of inhibition of H-RAS by H-rev107 are still unknown.

Results: Our results indicate that H-rev107 was co-precipitated with H-RAS and downregulated the levels of activated RAS (RAS-GTP) and ELK1-mediated transactivation in epidermal growth factor-stimulated and H-RAS-cotransfected HtTA cervical cancer cells. Furthermore, an acyl-biotin exchange assay demonstrated that H-rev107 reduced H-RAS palmitoylation. H-rev107 has been shown to be a PLA/AT that is involved in phospholipid metabolism. Treating cells with the PLA/AT inhibitor arachidonyl trifluoromethyl ketone (AACOCF3) or methyl arachidonyl fluorophosphate (MAFP) alleviated H-rev107-induced downregulation of the levels of acylated H-RAS. AACOCF3 and MAFP also increased activated RAS and ELK1-mediated transactivation in H-rev107-expressing HtTA cells following their treatment with epidermal growth factor. In contrast, treating cells with the acyl-protein thioesterase inhibitor palmostatin B enhanced H-rev107-mediated downregulation of acylated H-RAS in H-rev107-expressing cells. Palmostatin B had no effect on H-rev107-induced suppression of RAS-GTP levels or ELK1-mediated transactivation. These results suggest that H-rev107 decreases H-RAS activity through its PLA/AT activity to modulate H-RAS acylation.

Conclusions: We made the novel observation that H-rev107 decrease in the steady state levels of H-RAS palmitoylation through the phospholipase A/acyltransferase activity. H-rev107 is likely to suppress activation of the RAS signaling pathway by reducing the levels of palmitoylated H-RAS, which decreases the levels of GTP-bound H-RAS and also the activation of downstream molecules. Our study further suggests that the PLA/AT activity of H-rev107 may play an important role in H-rev107-mediated RAS suppression.

Show MeSH
Related in: MedlinePlus