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Nedd4-2-Mediated Ubiquitination Facilitates Processing of Surfactant Protein-C
Journal article   Open access   Peer reviewed

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

Juliana J. Conkright, Karen S. Apsley, Emily P Martin, Ross Ridsdale, Ward R. Rice, Cheng-Lun Na, Baoli Yang and Timothy E. Weaver
American Journal of Respiratory Cell and Molecular Biology, Vol.42(2), pp.181-189
02/01/2010
DOI: 10.1165/rcmb.2009-0058OC
PMCID: PMC2822980
PMID: 19423771
url
https://europepmc.org/articles/pmc2822980View
Published (Version of record) Open Access

Abstract

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.
 Obstetrics and Gynecology

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