TY - JOUR
T1 - Detection of the p110 β subunit of phosphatidylinositol 43-kinase complexed with neutral endopeptidase
AU - Shen, Ruoqian
AU - Milowsky, Matthew I.
AU - Ozaki, Naoko
AU - Navarro, Daniel
AU - Sumitomo, Makoto
AU - Xu, Yang
AU - Nanus, David M.
N1 - Copyright:
Copyright 2008 Elsevier B.V., All rights reserved.
PY - 2002/9
Y1 - 2002/9
N2 - 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. Materials and Methods: In this report, we investigated the association of NEP with cytoplasmic proteins using ProteinChip(R) 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 LNCaP cell lysates which had the identical molecular weight as the β-subunit of p110 subunit of phosphatidylinositol 3-kinase. The identity of the p110β was confirmed by Western blot analysis of NEP and p110β immunoprecipitates using monoclonal antibodies specific for NEP and p110β. Conclusion: These data confirm the association of phosphatidylinositol 3-kinase (consisting of the p85 adaptor and p110 β-subunit) with NEP. Furthermore, this work demonstrates the ability of mass spectrometry to identify proteins interacting with NEP and potentially other cell-surface peptidases.
AB - 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. Materials and Methods: In this report, we investigated the association of NEP with cytoplasmic proteins using ProteinChip(R) 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 LNCaP cell lysates which had the identical molecular weight as the β-subunit of p110 subunit of phosphatidylinositol 3-kinase. The identity of the p110β was confirmed by Western blot analysis of NEP and p110β immunoprecipitates using monoclonal antibodies specific for NEP and p110β. Conclusion: These data confirm the association of phosphatidylinositol 3-kinase (consisting of the p85 adaptor and p110 β-subunit) with NEP. Furthermore, this work demonstrates the ability of mass spectrometry to identify proteins interacting with NEP and potentially other cell-surface peptidases.
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M3 - Article
C2 - 12529960
AN - SCOPUS:0036765805
VL - 22
SP - 2533
EP - 2538
JO - Anticancer Research
JF - Anticancer Research
SN - 0250-7005
IS - 5
ER -