Domains Necessary for G alpha sub(12) Binding and Stimulation of Protein Phosphatase-2A (PP2A): Is G alpha sub(12) a Novel Regulatory Subunit of PP2A?

Many cellular signaling pathways share regulation by protein phosphatase-2A (PP2A), a widely expressed serine/threonine phosphatase, and the heterotrimeric G protein G alpha sub(12). PP2A activity is altered in carcinogenesis and in some neurodegenerative diseases. We have identified binding of G alpha sub(12) with the A alpha subunit of PP2A, a trimeric enzyme composed of A (scaffolding), B (regulatory), and C (catalytic) subunits and demonstrated that G alpha sub(12) stimulated phosphatase activity (J Biol Chem 279: 54983-54986, 2004). We now show in substrate-velocity analysis using purified PP2A that V sub(max) was stimulated 3- to 4-fold by glutathione transferase (GST)-G alpha sub(12) with little effect on K sub(m) values. To identify the binding domains mediating the A alpha -G alpha sub(12) interaction, an extensive mutational analysis was performed. Well-characterized mutations of A alpha were expressed in vitro and tested for binding to GST-G alpha sub(12) in pull-down assays. G alpha sub(12) binds to A alpha along repeats 7 to 10, and PP2A B subunits are not necessary for binding. To identify where A alpha binds to G alpha sub(12), a series of 61 G alpha sub(12) mutants were engineered to contain the sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS) in place of 6 consecutive amino acids. Mutant G alpha sub(12) proteins were individually expressed in human embryonic kidney cells and analyzed for interaction with GST or GST-A alpha in pull-down assays. The A alpha binding sites were localized to regions near the N and C termini of G alpha sub(12). The expression of constitutively activated G alpha sub(12) (QL alpha sub(12)) 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 G alpha sub(12) and PP2A A alpha , a model describing the binding surfaces and potential mechanisms of G alpha sub(12)-mediated PP2A activation is presented.