Unveiling the Molecular Dance: Exploring MK2 Binding and Protein-Protein Interactions

Disciplines

Biochemistry | Molecular Biology

Abstract (300 words maximum)

Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) is a protein kinase that modulates expression at the translation level by regulating RNA-binding proteins. Activated via the p38 MAPK and can promote chronic inflammatory diseases. There are two isoforms of MK2 – a short and long one. However, little information is available about the function of the short variation. The research was conducted once a week for three hours in an advanced biochemistry laboratory functioning as a Course-based Undergraduate Research Experience (CURE) to identify a possible binding partner and function of MK2. The control plasmid was pGBT9, and the binding domain (BD) plasmid was pGBTK7. BD plasmids pGBKT7-53 (p-53), pGBKT7-LAM, and pGBKT-MK2 were transformed into the yeast strain Y190. The activation domain (AD) plasmids, pGADT7-T (T-antigen), was transformed into the yeast strain Y187. A yeast two-hybrid assay was performed by mating the plasmid containing strains Y190 and Y187. If the transformed proteins interact, the binding domain and activation domains of the GAL4 transcription factors come together and can activate reporter genes, one of which results in expression of a-galactosidase that is secreted and reacts with the substrate X-a-gal resulting in a blue product. p-53 interacts with the T-antigen are known interactors and was used as a positive control, and when plated on α-xGal plate, gave bright blue colonies. Laminin, however, does not interact with the T-antigen and was used as the negative control and did not give blue colonies as expected. Our protein of interest MK2 fused to the gal4 binding domain was tested for autoactivation and we found that it does not activate reporter genes as expected. These findings conclude that the yeast 2 hybrid assay is working and that we can now proceed with screening a cDNA library of activation domain fusion proteins to test for potential MK2 binding proteins.

Academic department under which the project should be listed

CSM - Chemistry and Biochemistry

Primary Investigator (PI) Name

Rajnish Singh

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Unveiling the Molecular Dance: Exploring MK2 Binding and Protein-Protein Interactions

Mitogen-Activated Protein Kinase-Activated Protein Kinase 2 (MK2) is a protein kinase that modulates expression at the translation level by regulating RNA-binding proteins. Activated via the p38 MAPK and can promote chronic inflammatory diseases. There are two isoforms of MK2 – a short and long one. However, little information is available about the function of the short variation. The research was conducted once a week for three hours in an advanced biochemistry laboratory functioning as a Course-based Undergraduate Research Experience (CURE) to identify a possible binding partner and function of MK2. The control plasmid was pGBT9, and the binding domain (BD) plasmid was pGBTK7. BD plasmids pGBKT7-53 (p-53), pGBKT7-LAM, and pGBKT-MK2 were transformed into the yeast strain Y190. The activation domain (AD) plasmids, pGADT7-T (T-antigen), was transformed into the yeast strain Y187. A yeast two-hybrid assay was performed by mating the plasmid containing strains Y190 and Y187. If the transformed proteins interact, the binding domain and activation domains of the GAL4 transcription factors come together and can activate reporter genes, one of which results in expression of a-galactosidase that is secreted and reacts with the substrate X-a-gal resulting in a blue product. p-53 interacts with the T-antigen are known interactors and was used as a positive control, and when plated on α-xGal plate, gave bright blue colonies. Laminin, however, does not interact with the T-antigen and was used as the negative control and did not give blue colonies as expected. Our protein of interest MK2 fused to the gal4 binding domain was tested for autoactivation and we found that it does not activate reporter genes as expected. These findings conclude that the yeast 2 hybrid assay is working and that we can now proceed with screening a cDNA library of activation domain fusion proteins to test for potential MK2 binding proteins.