Detection of the p110 beta subunit of phosphatidylinositol 3-kinase complexed with neutral endopeptidase.
BACKGROUND
Neutral endopeptidase 24.11 (NEP) is a cell-surface peptidase that inactivates a variety of neuropeptide substrates. In addition to catalytic activity, NEP can exert biological effects through protein-protein interactions. We previously reported that NEP directly associated with tyrosine-phosphorylated Lyn kinase, and with the p85 subunit of the phosphatidylinositol 3-kinase (PI3 kinase) resulting in an NEP-Lyn-PI3 kinase protein complex.


METHODS
In this report, we investigated the association of NEP with cytoplasmic proteins using ProteinChip Array, surface enhanced laser desorption/ionization (SELDI) technology combined with time-of-flight mass spectrometry, as well as immunoprecipitation and Western blottings.


RESULTS
Using immunocapture on the ProteinChip surface, we identified a 122 kDa protein which associates with NEP derived from LNCaP cell lysates which had the identical molecular weight as the beta-subunit of p110 subunit of phosphatidylinositol 3-kinase. The identity of the p110 beta was confirmed by Western blot analysis of NEP and p110 beta immunoprecipitates using monoclonal antibodies specific for NEP and p110 beta.


CONCLUSIONS
These data confirm the association of phosphatidylinositol 3-kinase (consisting of the p85 adaptor and p110 beta-subunit) with NEP. Furthermore, this work demonstrates the ability of mass spectrometry to identify proteins interacting with NEP and potentially other cell-surface peptidases.
Shen
Ruoqian
R
Laboratory of Urological Oncology, Department of Urology, Joan and Sanford I. Weill Medical College of Cornell University, 1300 York Avenue, E-300 (Box 23), New York, NY 10021, USA. rshen@med.cornell.edu
Milowsky
Matthew I
MI
Ozaki
Naoko
N
Navarro
Daniel
D
Sumitomo
Makoto
M
Xu
Yang
Y
Nanus
David M
DM
D015153:Blotting, Western; D006801:Humans; D008297:Male; D013058:Mass Spectrometry; D008545:Melanoma / Q000201:enzymology; D015260:Neprilysin / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D011233:Precipitin Tests; D011471:Prostatic Neoplasms / Q000201:enzymology; D040081:Protein Array Analysis; D017434:Protein Structure, Tertiary; D014407:Tumor Cells, Cultured
D019869:Phosphatidylinositol 3-Kinases; D015260:Neprilysin