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Research ArticleArticle

Domains Necessary for Gα12 Binding and Stimulation of Protein Phosphatase-2A (PP2A): Is Gα12 a Novel Regulatory Subunit of PP2A?

Deguang Zhu, Robert I. Tate, Ralf Ruediger, Thomas E. Meigs and Bradley M. Denker
Molecular Pharmacology May 2007, 71 (5) 1268-1276; DOI: https://doi.org/10.1124/mol.106.033555
Deguang Zhu
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Robert I. Tate
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Ralf Ruediger
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Thomas E. Meigs
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Bradley M. Denker
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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 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.

Footnotes

  • This work was supported by National Institute of General Medical Sciences grants GM55223 and National Institute of Diabetes and Digestive and Kidney Diseases Polycystic Kidney Disease Center P50-DK074030 (to B.M.D.), National Institutes of Health grant CA100869 (to T.E.M.), and University of North Carolina-Asheville Undergraduate Research Grant from the North Carolina Biotechnology Center (to R.I.T.).

  • Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.

  • doi:10.1124/mol.106.033555.

  • ABBREVIATIONS: RGS, regulator of G protein signaling; PP2A, protein phosphatase-2A; MDCK, Madin Darby canine kidney; dox, doxycycline; HEAT repeat, 39 amino acid motif named for huntingtin, elongation, A subunit, target of rapamycin; HEK, human embryonic kidney; GST, glutathione transferase; PAGE, polyacrylamide gel electrophoresis; TBST, Tris-buffered saline/Tween 20; GTPγS, guanosine 5[prime]-3-O-(thio)triphosphate; WT, wild type.

    • Received December 15, 2006.
    • Accepted February 15, 2007.
  • The American Society for Pharmacology and Experimental Therapeutics
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Molecular Pharmacology: 71 (5)
Molecular Pharmacology
Vol. 71, Issue 5
1 May 2007
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Research ArticleArticle

Domains Necessary for Gα12 Binding and Stimulation of Protein Phosphatase-2A (PP2A): Is Gα12 a Novel Regulatory Subunit of PP2A?

Deguang Zhu, Robert I. Tate, Ralf Ruediger, Thomas E. Meigs and Bradley M. Denker
Molecular Pharmacology May 1, 2007, 71 (5) 1268-1276; DOI: https://doi.org/10.1124/mol.106.033555

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Research ArticleArticle

Domains Necessary for Gα12 Binding and Stimulation of Protein Phosphatase-2A (PP2A): Is Gα12 a Novel Regulatory Subunit of PP2A?

Deguang Zhu, Robert I. Tate, Ralf Ruediger, Thomas E. Meigs and Bradley M. Denker
Molecular Pharmacology May 1, 2007, 71 (5) 1268-1276; DOI: https://doi.org/10.1124/mol.106.033555
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