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Published ahead of print on May 7, 2009
Am. J. Respir. Cell Mol. Biol. 2009, doi:10.1165/rcmb.2009-0058OC
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Submitted on February 12, 2009
Accepted on May 6, 2009

NEDD4-2 Mediated Ubiquitination Facilitates Processing of Surfactant Protein C

Juliana J Conkright1, Karen S Apsley2, Emily P Martin2, Ross Ridsdale2, Ward R Rice2, Cheng-Lun Na2, Baoli Yang3, and Timothy E Weaver4*

1 Scripps Florida Translational Research Institute, Jupiter, Florida, United States, 2 Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio, United States; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States, 3 Department of Obstetrics and Gynecology, University of Iowa, Carver College of Medicine, Iowa City, Iowa, United States, 4 Division of Pulmonary Biology, Cincinnati Children's Hospital Medical Center, 3333 Burnet Ave (ML 7029), Cincinnati, Ohio, 45229-3039, United States; Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, Ohio, United States

* To whom correspondence should be addressed. E-mail: tim.weaver{at}cchmc.org.

We previously proposed a model of surfactant protein C (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. In order 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 medias analyzed for secreted SP-C 24h post-infection. Mutation of S12PPDYS17 completely blocked secretion of SP-C. PPDY (PY motif) has previously been shown to bind WW domains of Nedd4-like, E3 ubiquitin ligases. Purified recombinant GST-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 of the propeptide, proSP-C was immunoprecipitated from transiently transfected HEK293 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 HEK293 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 • ubiquitin • type II cell






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