The insulin resistance associated with type II diabetes and the loss of growth control found in many cancers are thought to have arisen from selective defects in protein kinase mediated signal transduction pathways utilized by insulin and related growth factors. An understanding of the components that normally govern the regulation of these pathways is therefore required if new therapies are to be developed to cure these seemingly intractable diseases. It is known that 99.9% of all phosphorylation events regulated by insulin and related growth factors occur on serine and threonine residues, and of this number, 90% of all dephosphorylation events are controlled by only two phosphatases, protein phosphatase 1 (PP-1) and protein phosphatase 2A (PP-2A). Central roles for PP-1 and PP-2A have been established in a diverse range of cellular pathways, including glucose metabolism, muscle contractility, cell cycle, gene expression, and the transport of ions and nutrients into cells. The involvement of PP-1 and PP-2A in all of these important events raises the question of how can these enzymes regulate cellular processes independently and selectively? This is further apparent when one considers that both enzymes are expressed at micromolar concentrations in cells and share 49% homology with one another. The answer to this paradox is the discovery that the catalytic subunits of these enzymes are targeted to selected sites and substrates intracellularly by a set of distinct regulatory subunits. In the case of PP-1 about 10 of these subunits have been characterized, five inhibit the enzymatic activity of the phosphatase toward all substrates, whereas the others appear to target the catalytic subunit to specific substrates in vivo. Importantly, this small number of regulatory proteins cannot account for all of the intracellular actions of PP-1. To identify new PP-1 binding proteins Dr. Haystead and his colleagues have developed microcystin-biotin to affinity purify the entire cellular content of active forms of PP-1 and PP-2A holoenzymes. Further affinity purification allows them to separate proteins that specifically interact with the catalytic subunit of PP-1 from those that bind PP-2A. In this proposal they will use state of the art technology to systematically microsequence PP-1 binding proteins present in skeletal muscle, liver, and adipose tissue. The amino acid sequence of the affinity purified proteins will be analyzed to determine the binding sites for the catalytic subunit, and biochemical site directed mutagenesis studies will be utilized to characterize these sites. Two important goals will be achieved as a result of this work; first, the data base of known PP-1 regulatory subunits will be expanded; second, the motifs that encode for binding of the catalytic subunit of PP-1 will be identified.
Showing the most recent 10 out of 18 publications
