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Domains Necessary for G₁₂ Binding and Stimulation of Protein Phosphatase-2A (PP2A): Is G₁₂ a Novel Regulatory Subunit of PP2A?

Renal Division, Brigham and Women's Hospital, and Harvard Medical School, Boston, Massachusetts (B.M.D., D.Z.); Department of Biology, University of North Carolina at Asheville, Asheville, North Carolina (R.I.T., T.E.M.); and Department of Pathology, University of California at San Diego, La Jolla, California (R.R.)

Many cellular signaling pathways share regulation by protein phosphatase-2A (PP2A), a widely expressed serine/threonine phosphatase, and the heterotrimeric G protein G₁₂. PP2A activity is altered in carcinogenesis and in some neurodegenerative diseases. We have identified binding of G₁₂ with the A subunit of PP2A, a trimeric enzyme composed of A (scaffolding), B (regulatory), and C (catalytic) subunits and demonstrated that G₁₂ stimulated phosphatase activity (J Biol Chem 279: 54983–54986, 2004). We now show in substrate-velocity analysis using purified PP2A that V_max was stimulated 3- to 4-fold by glutathione transferase (GST)-G₁₂ with little effect on K_m values. To identify the binding domains mediating the A-G₁₂ interaction, an extensive mutational analysis was performed. Well-characterized mutations of A were expressed in vitro and tested for binding to GST-G₁₂ in pull-down assays. G₁₂ binds to A along repeats 7 to 10, and PP2A B subunits are not necessary for binding. To identify where A binds to G₁₂, a series of 61 G₁₂ mutants were engineered to contain the sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS) in place of 6 consecutive amino acids. Mutant G₁₂ proteins were individually expressed in human embryonic kidney cells and analyzed for interaction with GST or GST-A in pull-down assays. The A binding sites were localized to regions near the N and C termini of G₁₂. The expression of constitutively activated G₁₂ (QL₁₂) in Madin Darby canine kidney cells stimulated PP2A activity as determined by decreased phosphorylation of tyrosine 307 on the catalytic subunit. Based on crystal structures of G₁₂ and PP2A A, a model describing the binding surfaces and potential mechanisms of G₁₂-mediated PP2A activation is presented.

Address correspondence to: Dr. Bradley M. Denker, Brigham and Women's Hospital, Harvard Institutes of Medicine, 77 Avenue Louis Pasteur, Boston, MA 02115. E-mail: bdenker{at}rics.bwh.harvard.edu

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