The active PKR kinase domain (KD) phosphorylates the Ser51 residue of translation initiation factor eIF2?. The activation of PKR KD requires precise dimerization and autophosphorylation, including threonine-446 (T446) autophosphorylation in the activation loop. Then, the phospho-PKR (active) recognizes eIF2? and transfers the ?- phosphate of ATP to the Ser51 residue of eIF2?. We hypothesize that, in latent PKR, the activation loop collapses into the active-site cavity, and dimerization of the N-terminal double-stranded RNA binding domains of PKR would cause a precise conformational change of the C-terminal KD dimer. As a result, the activation loop residue T446 is phosphorylated by any of three mechanisms: trans-inter-dimer, cis-intra-dimer and trans- intra-dimer. We will dissect each of these mechanisms, and uncouple the role of dimerization and T446 phosphorylation, in particular, how dimerization promotes T446 autophosphorylation and how T446 autophosphorylation influences dimerization. Also, we will take an electron paramagnetic resonance (EPR) spectroscopy based approach to examine the movement of the Ser51 loop of a cysteine-less eIF2? in the apo-form, in the phosphorylated form, and with/without addition of PKR.

Public Health Relevance

Project Narrative: Protein kinase R is directly involved in anti-viral defense responses and anti-proliferative effects on cancer cells, this fundamental knowledge of PKR will be useful to design anti- viral and anti-proliferative modulators.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Academic Research Enhancement Awards (AREA) (R15)
Project #
1R15GM101575-01
Application #
8288483
Study Section
Molecular Genetics A Study Section (MGA)
Program Officer
Gerratana, Barbara
Project Start
2012-06-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2015-05-31
Support Year
1
Fiscal Year
2012
Total Cost
$334,541
Indirect Cost
$108,041
Name
University of Wisconsin Milwaukee
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
627906399
City
Milwaukee
State
WI
Country
United States
Zip Code
53201
Sathe, Leena; Bolinger, Cheryl; Mannan, M Amin-ul et al. (2015) Evidence That Base-pairing Interaction between Intron and mRNA Leader Sequences Inhibits Initiation of HAC1 mRNA Translation in Yeast. J Biol Chem 290:21821-32
Dey, Madhusudan; Mann, Brian Rick; Anshu, Ashish et al. (2014) Activation of protein kinase PKR requires dimerization-induced cis-phosphorylation within the activation loop. J Biol Chem 289:5747-57