Identification of phosphorylation sites in protein kinase A substrates using artificial neural networks and mass spectrometry.
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.
Hjerrild
Majbrit
M
Department of Clinical Biochemistry, Glostrup Hospital, Nordre Ringvej 57, DK-2600 Glostrup, Denmark. mh@dcb-glostrup.dk
Stensballe
Allan
A
Rasmussen
Thomas E
TE
Kofoed
Christine B
CB
Blom
Nikolaj
N
Sicheritz-Ponten
Thomas
T
Larsen
Martin R
MR
Brunak
Søren
S
Jensen
Ole N
ON
Gammeltoft
Steen
S
D000465:Algorithms; D000818:Animals; D001185:Artificial Intelligence; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000378:metabolism; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D003198:Computer Simulation; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D018398:Homeodomain Proteins / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D009419:Nerve Tissue Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000099266:Engrailed 2 Protein
D018797:Cell Cycle Proteins; D017868:Cyclic AMP-Dependent Protein Kinases; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; D009419:Nerve Tissue Proteins; D009687:Nuclear Proteins; D011505:Protein-Tyrosine Kinases; D000071701:Regulatory Factor X Transcription Factors; D000099266:Engrailed 2 Protein; C093524:RFX5 protein, human; C069766:necdin; C499349:WEE1 protein, human; C499348:Wee1 protein, mouse