Interaction between DNA-PKcs and AMP-activated Protein Kinase (AMPK)

Abstract

I: AMPK activated Protein Kinase (AMPK) knockdown by small interfering RNA induces apoptosis under UV stress condition in Human Colorectal Carcinoma cell line. Two systems are essential in humans for genome integrity, DNA repair and apoptosis. Cells that are defective in DNA repair tend to accumulate excess DNA damage. Cells defective in apoptosis tend to survive with excess DNA damage and thus allow DNA replication pass DNA damages, causing mutations leading to carcinogenesis. Human DNA-dependent protein kinase (DNA-PK) is a nuclear localized serine /threonine protein kinase that is composed of a large catalytic subunit, DNA-PKcs (approximately 470kDa) and a heterodimeric protein Ku. It is involved in the repair of DNA double strand break repair by non homologous end joining pathway. Besides that it also acts as a scaffold protein to aid the localization of other DNA repair proteins to the site of damage. It also plays a role in the stability of telomere and the prevention of chromosomal end fusion. We found that DNA-PKcs interacts with AMP activated Protein Kinase, gamma 1 subunit. This interaction is prominent in UV stress condition. This interaction is confirmed by yeast two hybrid assay and co-immunoprecipitation test. Under UV stress condition, these two proteins co-localize, which is confirmed by confocal microscopy. Cells become more apoptotic when AMPKγ-1 is knockdown by siRNA as confirmed by cell death assays. These results give clue in the development of anticancer drugs targeting AMPK. II: Human glioma cell line proficient in DNA-dependent Protein Kinase (DNA-PKcs) is sensitive to cell death under glucose depleted conditions. M059K and M059J are human glioma cell lines. The former one is DNA dependent protein kinase catalytic subunit (DNA-PKcs) proficient, while the latter one is DNA-PKcs deficient. We checked the sensitivity of these two cell lines under low glucose condition. Under low glucose condition, AMP activated protein kinase (AMPK) is activated. AMPK is a serine/threonine protein kinase. It is the energy sensor in the eukaryotic cell. It regulates energy balance by monitoring changes in the cellular concentrations of the nucleotides AMP and ATP. An increase in AMP concentration, indicating an energy deficit, activates AMPK and initiates ATP generating pathways and suppresses those involved in ATP consumption. Conversely, high ATP concentration, indicating energy sufficiency prevents activation of AMPK. Under glucose depleted condition, in the glioma cell line, there is considerable cell death. Human glioma cell line, M059K is more sensitive than M059J as shown by cell death assays. Under glucose depleted condition, AMPK is activated and phosporylated at Threonine 172 of AMPK alpha in a time dependent manner. This phosphorylation effect is more prominent in M059K cell than M059J. This shows that DNA-PKcs, a DNA repair protein, is involved in the activation of AMPK under glucose depleted condition and causes the cell death.