Galpha12 directly interacts with PP2A: evidence FOR Galpha12-stimulated PP2A phosphatase activity and dephosphorylation of microtubule-associated protein, tau.
The Galpha(12/13) family of heterotrimeric G proteins modulate multiple cellular processes including regulation of the actin cytoskeleton. Galpha(12/13) interact with several cytoskeletal/scaffolding proteins, and in a yeast two-hybrid screen with Galpha(12), we detected an interaction with the scaffolding subunit (Aalpha) of the Ser/Thr phosphatase, protein phosphatase 2A (PP2A). PP2A dephosphorylates multiple substrates including tau, a microtubule-associated protein that is hyperphosphorylated in neurofibrillary tangles. The interaction of Aalpha and Galpha(12) was confirmed by coimmunoprecipitation studies in transfected COS cells and by glutathione S-transferase (GST)-Galpha(12) pull-downs from cell lysates of primary neurons. The interaction was specific for Aalpha and Galpha(12) and was independent of Galpha(12) conformation. Endogenous Aalpha and Galpha(12) colocalized by immunofluorescent microscopy in Caco-2 cells and in neurons. In vitro reconstitution of GST-Galpha(12) or recombinant Galpha(12) with PP2A core enzyme resulted in approximately 300% stimulation of PP2A activity that was not detected with other Galpha subunits and was similar with GTPgammaS- and GDP-liganded Galpha(12). When tau and active kinase (Cdk5 and p25) were cotransfected in to COS cells, there was robust tau phosphorylation. Co-expression of wild type or QLalpha(12) with tau and the active kinase resulted in 60 +/- 15% reductions in tau phosphorylation. In primary cortical neurons stimulated with lysophosphatitic acid, a 50% decrease in tau phosphorylation was observed. The Galpha(12) effect on tau phosphorylation was inhibited by the PP2A inhibitor, okadaic acid (50 nm), in COS cells and neurons. Taken together, these findings reveal novel, direct regulation of PP2A activity by Galpha(12) and potential in vivo modulation of PP2A target proteins including tau.
Zhu
Deguang
D
Renal Division, Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 77 Avenue Louis Pasteur, Boston, MA 02115, USA.
Kosik
Kenneth S
KS
Meigs
Thomas E
TE
Yanamadala
Vijay
V
Denker
Bradley M
BM
D000199:Actins / Q000737:chemistry; D000818:Animals; D015153:Blotting, Western; D019556:COS Cells; D018938:Caco-2 Cells; D003599:Cytoskeleton / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D044365:GTP-Binding Protein alpha Subunits, G12-G13 / Q000737:chemistry*; D049452:Green Fluorescent Proteins / Q000737:chemistry; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008024:Ligands; D008856:Microscopy, Fluorescence; D009474:Neurons / Q000378:metabolism; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D054648:Protein Phosphatase 2; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000737:chemistry; D015398:Signal Transduction; D014162:Transfection; D020798:Two-Hybrid System Techniques; D016875:tau Proteins / Q000378:metabolism
D000199:Actins; D018076:DNA, Complementary; D008024:Ligands; D011994:Recombinant Proteins; D016875:tau Proteins; D049452:Green Fluorescent Proteins; D019319:Okadaic Acid; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D044365:GTP-Binding Protein alpha Subunits, G12-G13