Identification of phosphorylation sites in protein kinase A substrates using artificial neural networks and mass spectrometry

J Proteome Res. May-Jun 2004;3(3):426-33. doi: 10.1021/pr0341033.

Abstract

Protein phosphorylation plays a key role in cell regulation and identification of phosphorylation sites is important for understanding their functional significance. Here, we present an artificial neural network algorithm: NetPhosK (http://www.cbs.dtu.dk/services/NetPhosK/) that predicts protein kinase A (PKA) phosphorylation sites. The neural network was trained with a positive set of 258 experimentally verified PKA phosphorylation sites. The predictions by NetPhosK were validated using four novel PKA substrates: Necdin, RFX5, En-2, and Wee 1. The four proteins were phosphorylated by PKA in vitro and 13 PKA phosphorylation sites were identified by mass spectrometry. NetPhosK was 100% sensitive and 41% specific in predicting PKA sites in the four proteins. These results demonstrate the potential of using integrated computational and experimental methods for detailed investigations of the phosphoproteome.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algorithms*
  • Animals
  • Artificial Intelligence*
  • COS Cells
  • Cell Cycle Proteins / metabolism
  • Chlorocebus aethiops
  • Cloning, Molecular
  • Computer Simulation
  • Cyclic AMP-Dependent Protein Kinases / metabolism*
  • DNA-Binding Proteins / metabolism*
  • Homeodomain Proteins / metabolism
  • Humans
  • Immunoprecipitation
  • Mice
  • Nerve Tissue Proteins / metabolism*
  • Nuclear Proteins / metabolism*
  • Phosphorylation
  • Protein-Tyrosine Kinases / metabolism
  • Regulatory Factor X Transcription Factors
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization

Substances

  • Cell Cycle Proteins
  • DNA-Binding Proteins
  • Homeodomain Proteins
  • Nerve Tissue Proteins
  • Nuclear Proteins
  • RFX5 protein, human
  • Regulatory Factor X Transcription Factors
  • engrailed 2 protein
  • necdin
  • Protein-Tyrosine Kinases
  • WEE1 protein, human
  • Wee1 protein, mouse
  • Cyclic AMP-Dependent Protein Kinases