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High-Throughput In Vitro Identification of Direct MAPK/Erk Substrates
Rona Grossman1, Ze'ev Paroush2
1Department of Developmental Biology and Cancer Research, Institute for Medical Research Israel-Canada (IMRIC), Faculty of Medicine, The Hebrew University, Jerusalem, 9112102, Israel.
Methods in Molecular Biology (Clifton, N.J.)
|December 8, 2016
Summary
This study introduces a high-throughput proteomics method to identify new nuclear targets of the Drosophila MAPK/Erk pathway. This approach aids in understanding cellular responses to kinase activity and signaling pathways.
Area of Science:
- Cellular biology
- Molecular biology
- Proteomics
Background:
- Protein phosphorylation by kinases regulates critical cellular processes like cell division, metabolism, and differentiation.
- Signaling pathways use sequential phosphorylation to transmit signals, altering gene expression and cell fate.
- Identifying direct kinase targets is crucial for understanding cellular responses to signaling pathway activation.
Purpose of the Study:
- To develop a high-throughput, genome-wide proteomics approach for discovering novel nuclear targets of the Drosophila MAPK/Erk pathway.
- To provide a method for identifying kinase substrates within nuclear compartments.
Main Methods:
- Utilized in vitro transcription and translation of cDNA pools with [35S]Methionine to create radiolabeled protein libraries.
- Performed biochemical kinase assays using recombinant, active Erk2 to phosphorylate protein pools.
- Detected potential MAPK/Erk substrates via shifted mobility on SDS-PAGE gels after phosphorylation.
Main Results:
- Successfully developed and applied a high-throughput proteomics approach to identify potential nuclear substrates of Drosophila MAPK/Erk.
- Demonstrated the detection of phosphorylated proteins indicative of kinase activity.
Conclusions:
- The described proteomics protocol is effective for uncovering novel nuclear targets of specific kinases, such as Drosophila MAPK/Erk.
- This method is adaptable for identifying targets of other kinases where in vitro phosphorylation assays are feasible.
