TY - JOUR T1 - Domains Necessary for Gα<sub>12</sub> Binding and Stimulation of Protein Phosphatase-2A (PP2A): Is Gα<sub>12</sub> a Novel Regulatory Subunit of PP2A? JF - Molecular Pharmacology JO - Mol Pharmacol SP - 1268 LP - 1276 DO - 10.1124/mol.106.033555 VL - 71 IS - 5 AU - Deguang Zhu AU - Robert I. Tate AU - Ralf Ruediger AU - Thomas E. Meigs AU - Bradley M. Denker Y1 - 2007/05/01 UR - http://molpharm.aspetjournals.org/content/71/5/1268.abstract N2 - Many cellular signaling pathways share regulation by protein phosphatase-2A (PP2A), a widely expressed serine/threonine phosphatase, and the heterotrimeric G protein Gα12. PP2A activity is altered in carcinogenesis and in some neurodegenerative diseases. We have identified binding of Gα12 with the Aα subunit of PP2A, a trimeric enzyme composed of A (scaffolding), B (regulatory), and C (catalytic) subunits and demonstrated that Gα12 stimulated phosphatase activity (J Biol Chem279: 54983–54986, 2004). We now show in substrate-velocity analysis using purified PP2A that Vmax was stimulated 3- to 4-fold by glutathione transferase (GST)-Gα12 with little effect on Km values. To identify the binding domains mediating the Aα-Gα12 interaction, an extensive mutational analysis was performed. Well-characterized mutations of Aα were expressed in vitro and tested for binding to GST-Gα12 in pull-down assays. Gα12 binds to Aα along repeats 7 to 10, and PP2A B subunits are not necessary for binding. To identify where Aα binds to Gα12, a series of 61 Gα12 mutants were engineered to contain the sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS) in place of 6 consecutive amino acids. Mutant Gα12 proteins were individually expressed in human embryonic kidney cells and analyzed for interaction with GST or GST-Aα in pull-down assays. The Aα binding sites were localized to regions near the N and C termini of Gα12. The expression of constitutively activated Gα12 (QLα12) 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 Gα12 and PP2A Aα, a model describing the binding surfaces and potential mechanisms of Gα12-mediated PP2A activation is presented. The American Society for Pharmacology and Experimental Therapeutics ER -