Rapamycin is a macrolide immunosuppressant that inhibits T cell proliferation and B cell immunoglobulin production. It blocks protein synthesis and arrests growth of cells, including yeast, in Gl phase. Potential clinical applications include attenuation of graft vs. host response in organ transplantation and treatment of autoimmune diseases and certain cancers. The goal of this research is to elucidate the rapamycin-sensitive pathway for activating translation through the action of unusual protein phosphatases. Rapamycin binds to an intracellular receptor protein called FKBP, and the drug-protein complex inhibits the protein kinase called target of rapamycin (TOR) or FRAP. Genetic analysis in yeast identified a Tap42 protein that co- immunoprecipitated with yeast protein phosphatases Sit4 and Pph21, the yeast versions of mammalian PP6 and PP2A. Rapamycin prevented binding of Tap42 to the phosphatases, but this did not occur in strains mutated in TOR, showing that Tap42 is downstream of TOR in the signaling pathway. Surprisingly, a mouse protein called alpha-4 is related in sequence to Tap42 and was discovered independently as a phosphoprotein associated with the B-cell receptor Ig-alpha protein. Preliminary studies show that murine alpha-4 binds purified human PP2A, displaces the other regulatory subunits, and changes substrate specificity. Epitope tagged alpha-4 expressed in COS cells co-immunoprecipitated with PP2A and caused dephosphorylation of the elongation factor EF2, without effects on PHAS-1 (eIF4E-BP1) or p70S6K, that also operate downstream of TOR.
The specific aims of this project are to: 1) define the structural features of alpha-4 and PP2A/PP6 required for binding, using truncated and mutated recombinant fusion proteins and epitope-tagged proteins in pull-down and co-precipitation assays. Produce mutant forms of alpha-4 that will not bind phosphatases to act as dominant negatives. 2) determine the kinetics and substrate specificity of alpha-4: PP2A relative to the AC dimer in biochemical assays, using defined substrates such as EF2, EF2 kinase, phosphorylase, MBP and peptides. 3) express dominant-negative forms of alpha-4 in fibroblasts and Jurkat T and Raji B cells and measure the phosphorylation of eEF2, initiation factors, kinases, as well as entry into S phase and sensitivity to rapamycin. 4) discover proteins that associate with the highly conserved N and C terminal domains of alpha-4, outside of the regions that bind to phosphatases. This will reveal the basis for substrate specificity and targeting of the alpha-4: phosphatase and possibly a site for association of alpha-4 with Ig-alpha. This project will discover new molecular mechanisms for control of protein synthesis and cell growth that are sensitive to rapamycin.
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