Phosphorylation of mRNA Decapping Protein Dcp1a by the ERK Signaling Pathway during Early Differentiation of 3T3-L1 Preadipocytes

abstract: ...Background Turnover of mRNA is a critical step in the regulation of gene expression, and an important step in mRNA decay is removal of the 5 cap. We previously demonstrated that the expression of some immediate early gene mRNAs is controlled by RNA stability during early differentiation of 3T3-L1 preadipocytes. Methodology/Principal Findings Here we show that the mouse decapping protein Dcp1a is phosphorylated via the ERK signaling pathway during early differentiation of preadipocytes. Mass spectrometry analysis and site-directed mutagenesis combined with a kinase assay identified ERK pathwaymediated dual phosphorylation at Ser 315 and Ser 319 of Dcp1a. To understand the functional effects of Dcp1a phosphorylation, we examined protein-protein interactions between Dcp1a and other decapping components with co-immunoprecipitation. Dcp1a interacted with Ddx6 and Edc3 through its proline-rich C-terminal extension, whereas the conserved EVH1 (enabled vasodilator-stimulated protein homology 1) domain in the N terminus of Dcp1a showed a stronger interaction with Dcp2. Once ERK signaling was activated, the interaction between Dcp1a and Ddx6, Edc3, or Edc4 was not affected by Dcp1a phosphorylation. Phosphorylated Dcp1a did, however, enhanced interaction with Dcp2. Protein complexes immunoprecipitated with the recombinant phosphomimetic Dcp1a(S315D/S319D) mutant contained more Dcp2 than did those immunoprecipitated with the nonphosphorylated Dcp1a(S315A/S319A) mutant. In addition, Dcp1a associated with AU-rich element (ARE)-containing mRNAs such as MAPK phosphatase-1 (MKP-1), whose mRNA stability was analyzed under the overexpression of Dcp1a constructs in the Dcp1a knockdown 3T3-L1 cells. Conclusions/Significance Our findings suggest that ERK-phosphorylated Dcp1a enhances its interaction with the decapping enzyme Dcp2 during early differentiation of 3T3-L1 cells.

... signaling was activated, the interaction between Dcp1a and Ddx6, Edc3, or Edc4 was not affected by Dcp1a phosphorylation. Phosphorylated Dcp1a did, however, enhanced interaction with Dcp2. Protein complexes immunoprecipitated with the recombinant phosphomimetic Dcp1a(S315D/S319D) mutant contained more Dcp2 than did those immunoprecipitated with the nonphosphorylated Dcp1a(S315A/S319A) mutant. In addition, Dcp1a associated with AU-rich element (ARE)-containing mRNAs such as MAPK phosphatase-1 (MKP-1

... similar interaction has not been observed in human cells, but Hedls/Ge-1/Edc4 is believed to be the essential factor responsible for the assembly of the decapping complex [16] [18] . Recent kinetic and structural studies of yeast decapping complexes have provided clues to how Dcp2p activity is regulated by Dcp1p [19] , [20] . Mammalian Dcp1a has little homology with yeast Dcp1p, and little is known about the details of the mammalian decapping complex assembly or the regulation of decapping activity

... isolated, and IP was performed as described in (C). The left panels in (C) and (D) represent 10% of the input, and the right panels represent immunoprecipitated complexes. One representative of three independent experiments with similar results is shown. Phosphorylation-independent association between Dcp1a and Ddx6, Edc3, and Edc4 We further examined whether ERK-mediated Dcp1a phosphorylation modulates interactions between Dcp1a and other decapping components. Flag-tagged Dcp1a was ectopically expressed together with HA-tagged CA or DN MAPKK1 mutants in HEK 293T cells. After IP using M2 beads, protein complexes were detected with western blotting using anti-Ddx6, anti-Edc3, or anti-Edc4. Interactions between Dcp1a and Ddx6, Edc3, and Edc4 were not affected by the phosphorylation status of Dcp1a ( Fig. 5A ). Endogenous protein interactions were demonstrated by co-IP with extracts from control or 1-h differentiation-induced 3T3-L1 cells in the presence or absence of U0126. Protein complexes immunoprecipitated by anti-Dcp1a contained Ddx6, Edc3, and Edc4, but not HuR (negative control) ( Fig. 5B ). No significant differences were observed in the protein levels immunoprecipitated by Dcp1a with different phosphorylation states. Together, these results suggested that the phosphorylation of Dcp1a did not affect its association with Edc3, Edc4, or Ddx6. Moreover, immunofluorescence staining showed that phosphorylation of Dcp1a did not alter its subcellular localization or P-body formation in HeLa cells

...-Edc4. The arrowhead indicates the specific Edc3 band. (B) Co-IP of Dcp1a and other decapping factors in 3T3-L1 cell extracts. Cell extracts from control cells or cells induced to differentiate for 1 h in the presence or absence of 20 M U0126 were incubated with anti-Dcp1a and protein ASepharose. After extensive washing, the precipitated protein complexes were analyzed with western blotting with anti-Dcp1a, anti-Ddx6, anti-Edc3, anti-Edc4, and anti-HuR. The arrowhead indicates the specific Ddx6 band. Similar results were independently reproduced three times. Phosphorylation of Dcp1a increases its association with Dcp2 Furthermore, we overexpressed Dcp1a and Dcp2 with different tags to perform co-IP experiments. Flag-tagged Dcp2 immunoprecipitated a greater amount of CA MAPKK1 induced phosphorylation of GFP-tagged Dcp1a in HEK 293T cells than did DN MAPKK1. This increased level was downregulated following treatment with U0126, whereas a similar amount of Edc4 was co-immunoprecipitated by Dcp2 in

infinome is a free SNP search engine. Link your 23andMe account for personalized reports.

Gene: EDC4 - enhancer of mRNA decapping 4 - (NCBI)

Images from Open Access Papers

Association Studies

Search Results from Open Access Papers