Detection of the p110 β subunit of phosphatidylinositol 43-kinase complexed with neutral endopeptidase

Ruoqian Shen, Matthew I. Milowsky, Naoko Ozaki, Daniel Navarro, Makoto Sumitomo, Yang Xu, David M. Nanus

Research output: Contribution to journalArticlepeer-review

8 Citations (Scopus)

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(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.

Original languageEnglish
Pages (from-to)2533-2538
Number of pages6
JournalAnticancer research
Volume22
Issue number5
Publication statusPublished - 09-2002
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Oncology
  • Cancer Research

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