Calmodulin Antagonizes a Calcium-Activated SCF Ubiquitin E3 Ligase Subunit, FBXL2, To Regulate Surfactant Homeostasis

Abstract

Calmodulin is a universal calcium-sensing protein that has pleiotropic effects. Here we show that calmodulin inhibits a new SCF (Skp1–Cullin–F-box) E3 ligase component, FBXL2. During Pseudomonas aeruginosa infection, SCF (FBXL2) targets the key enzyme, CCTα, for its monoubiquitination and degradation, thereby reducing synthesis of the indispensable membrane and surfactant component, phosphatidylcholine. P. aeruginosa triggers calcium influx and calcium-dependent activation of FBXL2 within the Golgi complex, where it engages CCTα. FBXL2 through its C terminus binds to the CCTα IQ motif. FBXL2 knockdown increases CCTα levels and phospholipid synthesis. The molecular interaction of FBXL2 with CCTα is opposed by calmodulin, which traffics to the Golgi complex, binds FBXL2 (residues 80 to 90) via its C terminus, and vies with the ligase for occupancy within the IQ motif. These observations were recapitulated in murine models of P. aeruginosa-induced surfactant deficiency, where calmodulin gene transfer reduced FBXL2 actions by stabilizing CCTα and lessening the severity of inflammatory lung injury. The results provide a unique model of calcium-regulated intermolecular competition between an E3 ligase subunit and an antagonist that is critically relevant to pneumonia and lipid homeostasis.

ACKNOWLEDGMENTS

We thank David Price and Robert Piper for critical review of the manuscript and helpful suggestions. We thank Michael Feldkamp for analysis of the calmodulin crystal structure. We specially thank Jessica C. Sieren and Geoffrey McLennan for their help on the micro-CT scan study.

This material is based upon work supported, in part, by the Department of Veterans Affairs, Veterans Health Administration, Office of Research and Development, Biomedical Laboratory Research and Development. This work was supported by a Merit Review Award from the Department of Veterans Affairs and NIH R01 grants HL081784, HL096376, HL097376, and HL098174 (to R.K.M.).

The contents of this report do not represent the views of the Department of Veterans Affairs or the U.S. Government.