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    A Comparative DNA Binding Study of the Human MAPK ERK2 and the Plant MAPK MPK4

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    Name:
    Master's Thesis_Siba Alharbi_Summer 2020_Final.pdf
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    3.363Mb
    Format:
    PDF
    Description:
    MS Thesis
    Embargo End Date:
    2021-07-28
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    Type
    Thesis
    Authors
    Alharbi, Siba I. cc
    Advisors
    Arold, Stefan T. cc
    Committee members
    Hirt, Heribert cc
    Al-Babili, Salim cc
    Program
    Bioscience
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Date
    2020-07
    Embargo End Date
    2021-07-28
    Permanent link to this record
    http://hdl.handle.net/10754/664480
    
    Metadata
    Show full item record
    Access Restrictions
    At the time of archiving, the student author of this thesis opted to temporarily restrict access to it. The full text of this thesis will become available to the public after the expiration of the embargo on 2021-07-28.
    Abstract
    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.
    Citation
    Alharbi, S. I. (2020). A Comparative DNA Binding Study of the Human MAPK ERK2 and the Plant MAPK MPK4. KAUST Research Repository. https://doi.org/10.25781/KAUST-752CI
    DOI
    10.25781/KAUST-752CI
    ae974a485f413a2113503eed53cd6c53
    10.25781/KAUST-752CI
    Scopus Count
    Collections
    Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program; Theses

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