Detection of the p110 beta subunit of phosphatidylinositol 3-kinase complexed with neutral endopeptidase.

Abstract:

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 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. CONCLUSION: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.

journal_name

Anticancer Res

journal_title

Anticancer research

authors

Shen R,Milowsky MI,Ozaki N,Navarro D,Sumitomo M,Xu Y,Nanus DM

subject

Has Abstract

pub_date

2002-09-01 00:00:00

pages

2533-8

issue

5

eissn

0250-7005

issn

1791-7530

journal_volume

22

pub_type

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