Mitogen-activated protein kinases (MAPKs) are an important subfamily of protein kinases that are well conserved in all eukaryotes. MAPKs are the final component of a three-tiered signaling module that regulates the activation of various essential cellular responses. They activate most of their substrates through catalyzing their phosphorylation. However, emerging evidence reveals that some MAPKs also possess non-catalytic functions. In particular, the human MAPK ERK2 can bind to DNA directly and mediate gene expression. The mechanism by which ERK2 binds to DNA is still unclear. In this work, we combined structural, biophysical and biochemical methods to confirm DNA binding by ERK2 and to investigate whether ERK2’s closest plant homolog MPK4 also binds to DNA. First, we identified a possible ERK2-like DNA consensus motif in plant MAPKs. We found that several plant MAPKs, including MPK4, harbor a basic motif (KARK/R or ARR/K) in a region corresponding to the ERK2 KAR motif reported to mediate DNA binding. Next, we determined the DNA binding affinity of ERK2 and MPK4 to different DNA fragments and found that MPK4 associated directly with DNA in vitro, albeit with a significantly lower affinity than did ERK2. Moreover, we observed that ERK2 and MPK4 showed preferred binding to different DNA sequences. Site-directed mutagenesis on the proposed DNA binding region of MPK4 greatly weakened DNA binding, confirming that MPK4 and ERK2 use the same structural elements to associate with DNA. Phosphorylation of the MAPKs through an upstream MKK affected the DNA binding capacity for both ERK2 and MPK4, although the effects differed. Lastly, we observed that a MPK4 mutant with a constitutively increased catalytic affinity displayed a markedly stronger DNA binding affinity compared to wild type MPK4 and phosphorylated MPK4. By demonstrating that the plant MPK4 associated with DNA in vitro, and that this association can be modified by phosphorylation and mutations, we open the possibility of additional kinase-independent functions in plant MAPKs.
|Date of Award||Jul 2020|
|Original language||English (US)|
- Biological, Environmental Science and Engineering
|Supervisor||Stefan Arold (Supervisor)|