Received for publication December 15, 2006.
Revised February 13, 2007.
Accepted for publication February 15, 2007.

Domains Necessary for G12 Binding and Stimulation of PP2A: is G 12 a Novel Regulatory Subunit of PP2A?

Abstract

Many cellular signaling pathways share regulation by 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 previously 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 G12 stimulated phosphatase activity (Zhu et al., 2004 JBC 279; 54983). We now show in substrate-velocity analysis using purified PP2A that V_max was stimulated 3-4 fold by GST-G12 with little effect on K_m. 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 7-10, 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 HEK cells, and analyzed for interaction with GST or GST-A in pull-downs assays. The Abinding sites were localized to regions near the N and C terminus of G12. The expression of constitutively activated G12 (QL12) in MDCK cells stimulated PP2A activity as determined by decreased phosphorylation of tyrosine-307 on the catalytic subunit. Based upon crystal structures of G12 and PP2A A, a model describing the binding surfaces and potential mechanisms of G12-mediated PP2A activation is presented.

Key words: G12,13;other G's, Protein Phosphatases (other), Structure-activity relationships and modeling, Mutagenesis/Chimeric approaches

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