Background - MAP (mitogen-activated protein) kinases play a critical role in mitogenic signaling and the failure to down-regulate their activity can lead to the development of cancer. MAP kinase phosphatases (MKP) are a family of protein phosphatases that specifically dephosphorylate and down-regulate MAP kinases (MAPKs). MKP5 is a newly discovered member of the MKP family that appears to employ a novel mechanism for negatively regulating the activity of MAPKs. Our preliminary studies indicate that MKP5 contains a novel DNA-binding and dimerization (DBD) domain that specifically interacts with DNA at the consensus sequence TGACTCA recognized by the transcription factor AP1 and found within promoters of genes whose products are involved in mitogenic and oncogenic signaling. Given that AP1 is a direct target for activation by MAPKs, the binding of the MKP5 DBD domain to the AP1 consensus sequence could provide an alternative robust mechanism for negating the action of MAPKs on transcription factors such as AP1. Here, we propose to study the molecular mechanism of action of the DBD domain of MKP5. Objectives - The fact that MKP5 lacks a transactivation domain suggests that the binding of its DBD domain to the AP1 site in DNA could competitively block out AP1 and thus lead to repression of its transcriptional activity. Our hypothesis is that the DBD domain of MKP5 acts as a transcriptional repressor of genes containing the AP1 consensus sequence TGACTCA in their promoters. In an effort to test the above hypothesis at the molecular level, we propose the following specific aims: (i) To determine 3D structure and dynamics of MKP5 DBD domain in complex with AP1 oligo;(ii) To measure thermodynamics and kinetics of MKP5 DBD domain binding to AP1 oligo;and (iii) To analyze cellular consequences of MKP5 DBD domain interaction with AP1 site. Methods - Our studies will be carried out as follows: (i) 3D structure of MKP5 DBD domain in complex with AP1 oligo and protein dynamics will be determined using nuclear magnetic resonance;(ii) Thermodynamics and kinetics of binding of MKP5 DBD domain to AP1 oligo will be studied using isothermal titration calorimetry and stop-flow fluorescence;and (iii) Cellular consequences of MKP5 DBD domain interaction with AP1 site in DNA will be analyzed using chromatin immuno-precipitation and luciferase reporter gene assays. Relevance - Hyperactivation of MAPKs and AP1 as well as down-regulated expression of MKP5 is linked to large fractions of human cancer. The studies proposed above will thus not only help to expand our knowledge of the molecular mechanisms involved in cancer but may also lead to the development of a novel, less toxic and more effective therapy for the treatment of cancer.
Regulatory mechanisms are an important facet of biology for their failure can lead to fatal diseases including cancer. MKP5 is a key player in the human body that keeps the activity of a number of proteins involved in causing cancer under control. Our studies will set out to understand how MKP5 executes such a remarkable feat and how we can exploit this new knowledge to design novel drugs with less toxicity but more effectiveness for the treatment of cancer.
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