Integrin α5β1 regulates PP2A complex assembly through PDE4D in atherosclerosis
Sanguk Yun, … , David C. Pallas, Martin A. Schwartz
Sanguk Yun, … , David C. Pallas, Martin A. Schwartz
Published August 13, 2019
Citation Information: J Clin Invest. 2019;129(11):4863-4874. https://doi.org/10.1172/JCI127692.
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Research Article Cell biology Vascular biology

Integrin α5β1 regulates PP2A complex assembly through PDE4D in atherosclerosis

Abstract

Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin α5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site, S651. Active PDE then hydrolyzes antiinflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site of PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in atherosclerosis-prone regions of arteries and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55α as the factor recruiting PP2A to PDE4D5. The B55α-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also, unexpectedly, stabilized the PP2A-B55α complex. The integrin-regulated, proatherosclerotic transcription factor Yap was also dephosphorylated and activated through this pathway. PDE4D5 therefore mediated matrix-specific regulation of endothelial cell phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex that has other targets. We believe these results may have widespread consequences for the control of cell function by integrins.

Authors

Sanguk Yun, Rui Hu, Melanie E. Schwaemmle, Alexander N. Scherer, Zhenwu Zhuang, Anthony J. Koleske, David C. Pallas, Martin A. Schwartz

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Figure 6

Yap is a PP2A-B55α substrate.

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Yap is a PP2A-B55α substrate. (A) BAECs plated on FN or MG for 6 hours w...
(A) BAECs plated on FN or MG for 6 hours were exposed to OSS for 2 hours. Phosphorylation of Yap (S127) was assayed by Western blotting (n = 4). (B) HUVECs transfected with B55α siRNA or PDE4D siRNA were detached and replated on FN and then sheared for 2 hours. Lysates were analyzed by Western blotting for p-Yap (S127). n = 3 independent experiments. (C) PDE4D5-knockdown BAECs were reconstituted with WT or N-terminally deleted mutant (ΔN). Cells were replated on FN, subjected to OSS for 2 hours, and Yap S127 phosphorylation assayed as in A (n = 6). (D) BAECs expressing GFP-B55α were transfected with Flag-Yap and replated on FN or MG for 20 minutes. Yap was immunoprecipitated with Flag Ab and probed for B55α. n = 3 independent experiments. (E) In the in vitro phosphatase assay, the PP2A catalytic subunit was immunoprecipitated with the PP2A C subunit Ab (clone 1D6) and incubated for 30 minutes at 37°C with p-Yap in the presence of increasing amounts of purified B55α. Reaction mixtures were immunoblotted for p-Yap (S127) (n = 4). *P < 0.05, by 1-way ANOVA (A and E) and 2-tailed Student’s t test (B and C).