268 : Effect of dystonia-causing point mutation in PACT on activation of interferon-induced protein kinase PKR

Abstract

PKR or Protein Kinase R is an interferon-induced, double stranded RNA (dsRNA)-activated, eIF2 α kinase that is a key regulator of cellular stress response pathways. PACT (also known as PRKRA, and DYT16) activates PKR in response to oxidative stress, endoplasmic reticulum (ER) stress, and serum starvation. Prolonged PKR activation in response to cellular stress leads to cell death by apoptosis. A recessively inherited form of early-onset dystonia has been recently identified to be associated with a homozygous missense mutation P222L in PACT. Dystonia is a neurological disorder defined by an occurrence of sustained involuntary muscle contractions, often leading to disabling and abnormal postures. Affected members from three unrelated Brazilian families have this P222L mutation in PACT’s coding region. The main hypothesis of this research is that in DYT16 patients the mutant PACT protein alters the normal functioning of the stress-response pathway thus leading to the disease phenotypes. We compared the ability of wild type PACT and mutant P222L proteins to interact with and activate PKR using in vitro biochemical assays. In addition, we compared activities of P222L and wt PACT during ER stress conditions in lymphoblast lines isolated from homozygous dystonia patients and controls. Our results indicate that as compared to the wild type PACT, substitution mutant P222L activates PKR with slower kinetics albeit more robustly and for longer duration. In addition, the affinity of PACT–PACT and consequently PACT–PKR interaction is enhanced in vivo in dystonia lymphoblast lines, thereby leading to intensified PKR activation and consequently enhanced cellular death. The implication of these results on development of dystonia due to enhanced apoptosis is unclear at present, since occurrence of neuronal apoptosis during dystonia progression remains unexplored. Our results elucidate new mechanistic details of PKR regulation by PACT and shed new light on its impact on stress induced cellular apoptosis.