Mitogen-activated Protein Kinase p38 Regulates the Wnt/Cyclic GMP/Ca2+ Non-canonical Pathway

Li Ma,Hsien-Yu Wang

doi:10.1074/jbc.m702840200

Abstract

The non-canonical Wnt/cyclic GMP/Ca(2+)/NF-AT pathway operates via Frizzled-2, a member of the superfamily of G protein-coupled receptors. In scanning for signaling events downstream of the Frizzled-2/G alpha t2/PDE6 triad activated in response to Wnt5a, we observed a strong activation of the mitogen-activated protein kinase p38 in mouse F9 teratocarcinoma embryonal cells. The activation of p38 is essential for NF-AT transcriptional activation mediated via Frizzled2. Wnt5a-stimulated p38 activation was rapid, sensitive to pertussis toxin, to siRNA against either G alpha t2 or p38 alpha, and to the p38 inhibitor SB203580. Real-time analysis of intracellular cyclic GMP using the Cygnet2 biosensor revealed p38 to act at the level of cyclic GMP, upstream of the mobilization of intracellular Ca(2+). Fluorescence resonance energy transfer (FRET) imaging reveals the changes in cyclic GMP in response to Wnt5a predominate about the cell membrane, and likewise sensitive to either siRNA targeting p38 or to treatment with SB203580. Dishevelled is not required for Wnt5a activation of p38; siRNAs targeting Dishevelleds and expression of the Dishevelled antagonist Dapper-1 do not suppress the p38 response to Wnt5a stimulation. These novel results are the first to detail a Dishevelled-independent Wnt response, demonstrating a critical role of the mitogen-activated protein kinase p38 in regulating the Wnt non-canonical pathway.

Highlights

Activation of Frizzled-2 by Wnt5a leads to activation of the phosphatidylinositol pathway [11], activation of the cyclic GMP phosphodiesterase PDE6 [13, 16], and inhibition of protein kinase G (PKG)2 [17], which leads to Ca2ϩ mobilization [17]
Ca2ϩ imaging with Fura-2 dye in zebrafish embryos [13, 18], mouse F9 teratocarcinoma cells [17], and human embryonic stem cells in culture [17] has revealed Wnt5a-stimulated Ca2ϩ mobilization in this non-canonical pathway to be downstream of PKG
Our results indicate a central role of p38 mitogen-activated protein kinase (MAPK) in the Wnt5a/Frizzled-2/G␣t2/PDE6/cyclic GMP and Ca2ϩ mobilization pathway that regulates NF-AT

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—Mouse F9 teratocarcinoma cells were obtained from the ATCC collection (Manassas, VA). Cells were lysed in 250 ␮l of lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerolphosphate, 1 mM Na3VO4, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 200 ␮M phenylmethylsulfonyl fluoride) and the mixture was subjected to centrifugation (20,000 ϫ g for 10 min at 4 °C). Cells were lysed in 250 ␮l of lysis buffer (20 mM Tris, pH 7.5; 150 mM NaCl, 1 mM EDTA, 1% Triton, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerolphosphate, 1 mM Na3VO4, 10 ␮g/ml leupeptin, 10 ␮g/ml aprotinin, and 200 ␮M phenylmethylsulfonyl fluoride) and the lysates were centrifuged at 20,000 ϫ g for 10 min at 4 °C. The results were expressed as percentage of cyclic GMP measured in the “control” cells

Treatment of Cells with Antisense

RESULTS

Protein antisense

Protein siRNA

Methods