Regulation of protein phosphatase-1

• Main Author: Gibbons, Jennifer A
• Format: Dissertation
• Language: English
• Published: ProQuest Dissertations & Theses 01-01-2006
• Subjects: Biochemistry; Cellular biology
• ISBN: 9780549148241; 0549148248

Cellular functions of protein phosphatase-1 (PP1), a major eukaryotic serine/threonine phosphatase, are defined by the association of PP1 catalytic subunits with protein inhibitors and regulatory subunits. Human tissues express four PP1 isoforms, which differ in their sequences principally near the N- and C-termini. Many PP1 regulators share a consensus "RVxF" motif, which docks within a hydrophobic pocket on the surface of the PP1 catalytic subunit, and established a key role of this PP1-binding sequence in the function of PP1 regulators. In addition, there is evidence that each PP1 regulator has a preference for one PP1 isoform over the others. However, it is unclear whether or not these PP1 isoforms have unique functions. Therefore, I chose to study the regulation between PP1 isoforms and their regulators by examining PP1's RVxF binding pocket and its variable N- and C-termini. First, I mutated PP1's RVxF hydrophobic binding pocket and used in vitro biochemistry to examine differences in in vitro interaction with, and inhibition by regulatory proteins In turn, I examined regulators with mutated RVxF motifs to determine their binding to the PP1 mutants. These studies showed that this PP1 binding pocket is important for recognition of the different RVxF motifs that regulators contain, and that these different RVxF motifs are important for the regulators' varying binding strengths to PP1. In addition, to test the hypothesis that eukaryotic regulators target individual PP1 catalytic subunits to specific subcellular compartments to regulate phosphoproteins located at these sites, I expressed human PP1 isoforms in Saccharomyces cerevisiae, which possesses a single PP1 gene (GLC7) that is essential for yeast viability. These yeast strains were viable and had unique characteristics. Binding of the human PP1 isoforms to G1c7p regulators that are important for these yeast phenotypes was also studied. By combining yeast biology with in vitro biochemistry I was able to determine that human PP1 isoforms do have unique regulatory and binding abilities. In summary, this work has shown that PP1 regulation is affected by differential binding of regulatory proteins to its RVxF binding pocket and the interaction of each human PP1 isoform with these regulators.