Nedd4-2-mediated ubiquitination facilitates processing of surfactant protein-C.
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 S(12)PPDYS(17) 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 S(12)PPDYS(17)mutation 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.
Cincinnati Children's Hospital Medical Center, Division of Pulmonary Biology, University of Cincinnati College of Medicine, Cincinnati, Ohio, USA.
Juliana J
JJ
Conkright
Karen S
KS
Apsley
Emily P
EP
Martin
Ross
R
Ridsdale
Ward R
WR
Rice
Cheng-Lun
CL
Na
Baoli
B
Yang
Timothy E
TE
Weaver
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D002460:Cell Line; D056827:Endosomal Sorting Complexes Required for Transport / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D051379:Mice; D018345:Mice, Knockout; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D000075702:Nedd4 Ubiquitin Protein Ligases; D010455:Peptides / Q000737:chemistry / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D011498:Protein Precursors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary; D011650:Pulmonary Alveoli / Q000166:cytology / Q000378:metabolism*; D037721:Pulmonary Surfactant-Associated Protein C; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D054875:Ubiquitination
D056827:Endosomal Sorting Complexes Required for Transport; D036341:Intercellular Signaling Peptides and Proteins; D010455:Peptides; D011498:Protein Precursors; D037721:Pulmonary Surfactant-Associated Protein C; D011994:Recombinant Proteins; C489680:Sftpc protein, mouse; D000075702:Nedd4 Ubiquitin Protein Ligases; C000618483:Nedd4 protein, human; C000618485:Nedd4L protein, human; C000618461:Nedd4l protein, mouse; D044767:Ubiquitin-Protein Ligases