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.
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.
Authors: Sakeneh Zraika; Kathryn Aston-Mourney; Peter Marek; Rebecca L Hull; Pattie S Green; Jayalakshmi Udayasankar; Shoba L Subramanian; Daniel P Raleigh; Steven E Kahn Journal: J Biol Chem Date: 2010-04-16 Impact factor: 5.157