Consequences of loss of the murine dsRNA-dependent protein kinase, PKR, by natural mutation or genetic ablation.
|Title:||Consequences of loss of the murine dsRNA-dependent protein kinase, PKR, by natural mutation or genetic ablation.|
|Abstract:||The IFN-induced, dsRNA activated protein kinase PKR, is a member of the family of eIF-2 a protein kinases that has well defined roles in anti-viral, anti-proliferative and apoptosis responses. Using a strategy designed to identify phosphotyrosine protein kinases, we have identified PKR as a member of the family of dual specificity kinases since it phosphorylates residues on serine, threonine and autophosphorylates on tyrosine residues. Although this activity has not yet been identified in mammalian expression of PKR, the significance of this novel property raises the possibility of new signaling pathways impinged by PKR. This work examined the consequences of mutation of PKR, initially examining a mutation identified in a lymphocytic leukemia cell. The putative mPKR del polypeptide encoded by this mutation was cloned and tested in expression studies. Although mPKRdel was shown to be catalytically inactive and to bind dsRNA and endogenous PKR, it nonetheless did not demonstrate transdominant inhibition of PKR or alter cell growth when expressed. Instead, no evidence of expression of the mutant polypeptide could be found in the lymphocytic leukemia cell and it is believed the mutation is either silent or exerts an effect not evident in our assays. A more direct approach to the implications of loss of PKR function was taken using targeted disruption of PKR in the mouse by homologous recombination. Functional ablation of PKR was accomplished by targeting the catalytic domain, in contrast to others who had disrupted PKR at the amino-terminal end. The absence of elevated tumorigenesis, normal anti-vaccinia and influenza responses, intact apoptotic induction and unimpaired cytokine signaling was unexpected. Although possible that a subtle phenotype has been missed, the major finding of this work is that non-concordance in phenotype compared to the other PKR-null model suggests that loss of PKR can be compensated by previously unappreciated homologs. The unambiguous inactivation of PKR catalytic function has allowed us to determine that rescue of PKR ablation is occurring since phosphorylation of eIF-2 a is unaffected. Finally, PKR ablation appears to impair Epo signaling in erythroid CFU suggesting non-redundancy in these cells.|
|Collection||Thèses, 1910 - 2010 // Theses, 1910 - 2010|