Domains Necessary for G (cid:1) 12 Binding and Stimulation of Protein Phosphatase-2A (PP2A): Is G (cid:1) 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 (cid:1) 12 . PP2A activity is altered in carcinogenesis and in some neurodegenerative diseases. We have identified binding of G (cid:1) 12 with the A (cid:1) subunit of PP2A, a trimeric enzyme composed of A (scaffold- ing), B (regulatory), and C (catalytic) subunits and demonstrated that G (cid:1) 12 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)-G (cid:1) 12 with little effect on K m values. To identify the binding domains mediating the A (cid:1) -G (cid:1) 12 interaction, an extensive mutational analysis was performed. Well-characterized mutations of A (cid:1) were expressed in vitro and tested for binding to GST-G (cid:1) 12 in pull-down assays. G (cid:1) 12 binds to A (cid:1) along repeats 7 to 10, and PP2A B subunits are not necessary for binding. To (cid:1) to (cid:1) 12 , a series of 61 G (cid:1) 12 mutants were engineered to contain the sequence Asn-Ala-Ala-Ile-Arg-Ser (NAAIRS) in place of 6 consecutive amino acids. Mutant G (cid:1) 12 proteins were individually expressed in human embryonic kidney cells and analyzed for interaction with GST or GST-A (cid:1) in pull-down assays. The A (cid:1) binding sites were localized to regions near the N and C termini of G (cid:1) 12 . The expression of constitutively activated G (cid:1) 12 (QL (cid:1) 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 (cid:1) 12 and PP2A A (cid:1) , a model describing the binding surfaces and potential mechanisms of G (cid:1) 12 -mediated PP2A activation is presented.

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