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Nedd4-2–Mediated Ubiquitination Facilitates Processing of Surfactant Protein–C

¹ Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology and Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio; ² Scripps Florida Translational Research Institute, Jupiter, Florida; and ³ Department of Obstetrics and Gynecology, Carver College of Medicine, University of Iowa, Iowa City, Iowa

Correspondence and requests for reprints should be addressed to Timothy E. Weaver, Ph.D., Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology, MLC7029, 3333 Burnet Avenue, Cincinnati, OH 45229-3039. E-mail: tim.weaver{at}cchmc.org

We previously proposed a model of surfactant protein (SP)–C biosynthesis in which internalization of the proprotein from the limiting membrane of the multivesicular body to internal vesicles represents a key step in the processing and secretion of SP-C. To test this hypothesis, alanine mutagenesis of the N-terminal propeptide of SP-C was performed. Adenoviruses encoding mutant proproteins were infected into type II cells isolated from Sftpc^–/– mice, and media analyzed for secreted SP-C 24 hours after infection. Mutation of S¹²PPDYS¹⁷ completely blocked secretion of SP-C. PPDY (PY motif) has previously been shown to bind WW domains of neural precursor cell-expressed developmentally down-regulated (Nedd) 4-like E3 ubiquitin ligases. Purified recombinant glutathione S-transferase–SP-C propeptide (residues 1–35) bound recombinant Nedd4-2 strongly, and Nedd4 weakly; the S¹²PPDYS¹⁷mutation abrogated binding of SP-C to Nedd4-2. Immobilized recombinant Nedd4-2 WW domain captured SP-C proprotein from mouse type II cell lysates; in the reverse pulldown, endogenous SP-C in type II cells was captured by recombinant Nedd4-2. To determine if the interaction of Nedd4-2 and SP-C resulted in ubiquitination, the SP-C proprotein was immunoprecipitated from transiently transfected human embryonic kidney 293 cells, and analyzed by SDS-PAGE/Western blotting with ubiquitin antibody. Two ubiquitinated forms of SP-C were detected; ubiquitination was blocked by mutation of K6, but not K34, in the SP-C propeptide. Mutation of K6 also inhibited processing of SP-C proprotein to the mature peptide in human embryonic kidney 293 cells. Nedd4-2–mediated ubiquitination regulates lumenal relocation of SP-C, leading to processing and, ultimately, secretion of SP-C.

Key Words: E3 ligase • multivesicular body • PY motif • type II cell • ubiquitin

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