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Affinity purification of ARE-binding proteins identifies polyA-binding protein 1 as a potential substrate in
Frank Bollig1, Reinhard Winzen, Matthias Gaestel
1Institute of Pharmacology, Medical School Hannover, Germany.
Abstract:
An important determinant for the expression level of cytokines and proto-oncogenes is the rate of degradation of their mRNAs. AU-rich sequence elements (AREs) in the 3(') untranslated regions have been found to impose rapid decay of these mRNAs. ARE-containing mRNAs can be stabilized in response to external signals which activate the p38 MAP kinase cascade including the p38 MAP kinase substrate MAPKAP kinase 2 (MK2). In an attempt to identify components downstream of MK2 in this pathway we analyzed several proteins which selectively interact with the ARE of GM-CSF mRNA. One of them, the cytoplasmic poly(A)-binding protein PABP1, co-migrated with a protein that showed prominent phosphorylation by recombinant MK2. Phosphorylation by MK2 was confirmed using PABP1 purified by affinity chromatography on poly(A) RNA. The selective interaction with an ARE-containing RNA and the phosphorylation by MK2 suggest that PABP1 plays a regulatory role in ARE-dependent mRNA decay and its modulation by the p38 MAP kinase cascade.
Insights
Cytoplasmic poly(A)-binding protein PABP1 is phosphorylated by MAPKAP kinase 2 (MK2). This phosphorylation regulates AU-rich element-dependent mRNA decay via the p38 MAP kinase pathway, impacting gene expression.
Area of Science:
- Molecular Biology
- Cell Signaling
- Gene Expression Regulation
Background:
- Cytokine and proto-oncogene expression levels are influenced by mRNA degradation rates.
- AU-rich elements (AREs) in 3' untranslated regions mediate rapid mRNA decay.
- The p38 MAP kinase cascade, including MAPKAP kinase 2 (MK2), stabilizes ARE-containing mRNAs upon external signaling.
Purpose of the Study:
- To identify downstream components of MK2 in the p38 MAP kinase pathway.
- To investigate the role of proteins interacting with the ARE of GM-CSF mRNA.
Main Methods:
- Analysis of proteins interacting with the ARE of GM-CSF mRNA.
- Co-migration studies to identify proteins phosphorylated by recombinant MK2.
- Affinity chromatography using poly(A) RNA for protein purification.
- Confirmation of MK2-mediated phosphorylation of purified PABP1.
Main Results:
- Cytoplasmic poly(A)-binding protein PABP1 was identified as a protein interacting with the ARE.
- PABP1 co-migrated with a protein prominently phosphorylated by recombinant MK2.
- Purified PABP1 was confirmed to be phosphorylated by MK2.
Conclusions:
- PABP1 selectively interacts with ARE-containing RNA.
- MK2-mediated phosphorylation of PABP1 suggests a regulatory role in ARE-dependent mRNA decay.
- PABP1 is a key component modulated by the p38 MAP kinase cascade in mRNA stability.