Determination of Mitogen‐Activated Protein Kinase‐Activated Protein Kinase‐2 (MK2) Function Through a Human cDNA Library Screen of Possible Binding Proteins
Disciplines
Biochemistry | Cell Biology | Laboratory and Basic Science Research | Molecular Biology
Abstract (300 words maximum)
Protein interactions are essential to the regulation of living systems, constituting pathways that elicit specific responses. Activation of the p38 MAPK pathway by environmental stressors plays a significant role in cancer development and inflammation. The inhibition of Mitogen‐Activated Protein Kinase‐Activated Protein Kinase‐2 (MK2), a downstream substrate, has been shown to decrease the progression of tumors in vivo, making it a desirable drug target. Therefore, we intend to explore the functions of MK2-isoform 2 by screening for interacting proteins from a human cDNA library. This will be conducted via the Yeast Two-Hybrid System, which involves fusing the binding (BD) and activation domains (AD) of the Gal4 protein to various ‘‘bait’’ and ‘‘prey’’ proteins, respectively, within plasmids. The bait will then ‘‘fish’’ through prey proteins, and if a successful interaction occurs, the Gal4 domains will be brought into contact and reconstitute at the promoters for the His3, Ade2, Aur1-c, or Mel1 genes. Binding is visualized through Mel1 expression of α-galactosidase, an enzyme responsible for cleaving X-α-Gal into a blue product. After Maxiprep purification of MK2-BD, BD and AD control plasmids were separately transformed into the yeast strains Y190 and Y187 to evaluate system performance. Cells containing positive control plasmids that express p53 and T-antigen proteins were mated and then streaked onto X-α-Gal supplemented plates, accurately resulting in blue colonies. Moreover, white colonies were observed when T-antigen and Lamin negative control plasmids were mated. This was also done with MK2-BD by itself and with an empty pGAD vector, resulting in no blue colonies and indicating no autoactivation. With the time and resources available to our Course-Based Undergraduate Research Experience group (CHEM 3512L), we aim to next mate yeast containing the MK2-BD plasmid with those carrying a library of AD-prey proteins to detect potential interactors and further characterize this important, yet less studied kinase.
Academic department under which the project should be listed
CSM - Chemistry and Biochemistry
Primary Investigator (PI) Name
Rajnish Singh
Determination of Mitogen‐Activated Protein Kinase‐Activated Protein Kinase‐2 (MK2) Function Through a Human cDNA Library Screen of Possible Binding Proteins
Protein interactions are essential to the regulation of living systems, constituting pathways that elicit specific responses. Activation of the p38 MAPK pathway by environmental stressors plays a significant role in cancer development and inflammation. The inhibition of Mitogen‐Activated Protein Kinase‐Activated Protein Kinase‐2 (MK2), a downstream substrate, has been shown to decrease the progression of tumors in vivo, making it a desirable drug target. Therefore, we intend to explore the functions of MK2-isoform 2 by screening for interacting proteins from a human cDNA library. This will be conducted via the Yeast Two-Hybrid System, which involves fusing the binding (BD) and activation domains (AD) of the Gal4 protein to various ‘‘bait’’ and ‘‘prey’’ proteins, respectively, within plasmids. The bait will then ‘‘fish’’ through prey proteins, and if a successful interaction occurs, the Gal4 domains will be brought into contact and reconstitute at the promoters for the His3, Ade2, Aur1-c, or Mel1 genes. Binding is visualized through Mel1 expression of α-galactosidase, an enzyme responsible for cleaving X-α-Gal into a blue product. After Maxiprep purification of MK2-BD, BD and AD control plasmids were separately transformed into the yeast strains Y190 and Y187 to evaluate system performance. Cells containing positive control plasmids that express p53 and T-antigen proteins were mated and then streaked onto X-α-Gal supplemented plates, accurately resulting in blue colonies. Moreover, white colonies were observed when T-antigen and Lamin negative control plasmids were mated. This was also done with MK2-BD by itself and with an empty pGAD vector, resulting in no blue colonies and indicating no autoactivation. With the time and resources available to our Course-Based Undergraduate Research Experience group (CHEM 3512L), we aim to next mate yeast containing the MK2-BD plasmid with those carrying a library of AD-prey proteins to detect potential interactors and further characterize this important, yet less studied kinase.