Chaperone-mediated biogenesis and activity regulation of protein phosphatase-1

The synthesis of enzymes involves multiple, tightly regulated steps including transcription, translation, post-translational maturation, and the association of co-factors and regulatory subunits. Protein phosphatase-1 (PP1) is a very conserved and essential eukaryotic Ser/Thr phosphatase that functions as the catalytic subunit of at least 200 distinct holoenzymes. However, it is not understood how PP1 acquires its native conformation and catalytic-site metals. Neither is it known how PP1 forms holoenzymes through association with regulatory subunits that determine when and where the phosphatase acts.

Here, we propose to explore the role of two ancient PP1-interacting proteins, i.e. Sds22 and Inhibitor-3, as chaperones that guide the biogenesis and degradation of PP1. Specifically modified cell lines will be generated to study the role of Sds22 and Inhibitor-3 in the biogenesis and activity regulation of PP1. Additional regulators of PP1 biogenesis will be identified by trapping of immature forms of PP1 and by a novel technique that enables the tagging of proteins that interact with PP1/Sds22 or PP1/Inhibitor-3. In addition, we will use rabbit reticulocyte lysates to reconstitute the biogenesis of PP1. Collectively, these experiments are expected to generate key insights into the biogenesis and degradation of PP1.