Abstract

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 V_max was stimulated 3- to 4-fold by glutathione transferase (GST)-Gα₁₂ with little effect on K_m 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.