Protein phosphatase-1 (PP-1) is a critical regulator of several key cellular processes, including apoptosis. The phosphatase activity of the PP-1 catalytic subunit (PP-1cat) is regulated by phosphorylation and by association with regulatory proteins and targeting subunits. The major regulated form of PP-1 in brain is the PP-1I complex, made up of PP-1cat and its regulatory protein inhibitor-2 (I-2). PP-1 I is basally inactive and is regulated by kinases through phosphorylation of both I-2 and PP-1cat. Recently, PP-1I was found to be activated in pig brain in a model of global cerebral ischemia, and co-purified with 14-3-3γ and PFTAIRE kinase (PFTK1). This study was designed to elucidate the role of 14-3-3γ and PFTK1 in the PP-1I complex.
In phosphatase activity assays, 14-3-3γ inhibited dephosphorylation by PP-1cat in a substrate-dependent manner. Therefore we suggest that 14-3-3γ might regulate PP-1 signaling by antagonizing its biological function rather than directly binding as a regulatory protein. PFTK1 has yet to be isolated in active form; thus, human PFTK1 was immunoprecipitated from HEK cells and used as kinase in in vitro phosphorylation assays and PP-1I activity assays. PFTK1 phosphorylated I-2 at Ser86, a site known to potentiate PP-1I activation, and PP-1cat at Thr-320, a site known to inhibit catalytic activity.
These studies identify PFTK1 as a novel I-2 and PP-1cat kinase, and implicate PFTK1 in the regulation of PP-1 activity. In addition, these data constitute the first report on the stoichiometry of phosphorylation of a substrate by PFTK1. We propose that PFTK1 is a novel regulator of PP-1 I and PP-1cat activity, and suggest roles for the bidirectional regulation of PP-1 by PFTK1 in cell death signaling following cerebral ischemia and in the control of cell cycle progression.
Although cerebral ischemia is a deadly medical emergency with a worldwide scope, there is a paucity of efficacious treatment. This is due to the complex nature of ischemic stroke pathology and the failure of animal models to mimic clinical conditions. Due to the multifaceted pathology of cerebral ischemia, it is unrealistic to expect a single target agent to be effective in the treatment of ischemic stroke. PP-1 and PP-2A have multiple roles in the cellular changes triggered by ischemia and reperfusion in the brain. These functions are discussed, and PP-1 and PP-2A are suggested as promising pleiotropic targets for the development of novel therapies for ischemic stroke.
|Advisor:||Hemmings, Hugh C., Jr.|
|Commitee:||Buck, Jochen, Lee, Francis S., Levin, Lonny|
|School:||Weill Medical College of Cornell University|
|School Location:||United States -- New York|
|Source:||DAI-B 74/05(E), Dissertation Abstracts International|
|Subjects:||Pharmacology, Biochemistry, Organic chemistry|
|Keywords:||Cyclin-dependent kinases, PFTK1, Phosphorylation, Protein phosphatase-1, Regulatory protein inhibitor-2|
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