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Surfactant Protein C Interacts with WW Domains of Nedd 4 Via an N‐terminal PPDY Motif
Author(s) -
Kotorashvili Adam,
Mulugeta Surafel,
Russo Scott J.,
Beers Michael F.
Publication year - 2007
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.21.5.a651-d
Subject(s) - alanine scanning , fusion protein , transfection , surfactant protein c , protein–protein interaction , wild type , immunoprecipitation , mutant , chemistry , microbiology and biotechnology , biology , biochemistry , mutagenesis , recombinant dna , gene
Surfactant protein C (SP‐C) is a small hydrophobic protein synthesized exclusively in alveolar type 2 cells as a 21 kD propeptide (proSP‐C 21 ) and proteolytically processed to yield a 3.7 kD mature form. We previously reported that the conserved sequence M 10 ESPPDYST 18 within the NH 2 SP‐C propeptide is essential for its targeting to distal processing organelles. However, the molecular mechanisms mediating these events are incompletely defined. Scanning alanine mutagenesis and binding assays were used to further characterize proSP‐C 21 NH 2 region. In transfected A549 cells, an EGFP/wild‐type SP‐C fusion was targeted to lysosomal like organelles while substituting A for E 11 ‐T 18 or S 12 PPDY 16 were restricted to ER. Since PPxY sequences are known ligands for Type I WW protein domains, we sought to identify interactions of proSP‐C PPDY with WW domains. Using a solid phase binding WW domain array, a GST fusion construct containing only the proSP‐C N‐terminus showed strong binding to multiple WW domains in Nedd‐4 and Nedd 4‐like E3 ligases. Co‐immunoprecipitation of A549 cells transfected with EGFP/proSP‐C 21 fusions revealed wild type but not PPDY mutant proSP‐C binds to Nedd 4. A similar interaction was also observed in type 2 cells. These findings suggest that Nedd 4 plays a key role in proSP‐C subcellular trafficking via WW domain‐mediated interaction with a PPDY motif in the NH 2 propeptide. Supported by NIH 19737 (MFB) and HL 074064 (MFB;SM)