Systematic characterization of phosphorylation sites in NFATc2 by linear ion trap mass spectrometry.
Members of the nuclear factor of activated T cells (NFAT) family of transcription factors regulate transcription of genes involved in the function of many different cellular systems. Activity of NFAT proteins is regulated by a complex interplay of phosphorylation events that are still poorly understood. In this work, we take advantage of the high scanning speed of the linear ion trap to develop a method to make a systematic characterization of NFATc2 phosphorylation. The method is based on the simultaneous monitoring of all tryptic peptides that can be detected by MS and contain potential phosphorylation sites. By this approach, we detected six NFATc2 phosphorylation sites by c-Jun NH2-terminal kinase (JNK) in vitro in only one experiment; a further site was also identified by performing digestion in solution. Using this approach, we have also characterized five basal phosphorylation sites in NFATc2 protein expressed in HEK cell cultures. Two of these NFATc2 phosphorylation sites in vivo have not been described before. The simplicity and sensitivity of this technique, which can be applied to any potential modification, makes it particularly attractive for the systematic detection of post-translational modifications of specific target proteins both in vitro and in vivo.
Villar
Margarita
M
Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas, Universidad Autónoma de Madrid, Madrid, Spain.
Ortega-Pérez
Inmaculada
I
Were
Felipe
F
Cano
Eva
E
Redondo
Juan Miguel
JM
Vázquez
Jesús
J
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D050778:NFATC Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010455:Peptides / Q000737:chemistry / Q000235:genetics; D010766:Phosphorylation; D053719:Tandem Mass Spectrometry
D050778:NFATC Transcription Factors; C494007:Nfatc2 protein, mouse; D010455:Peptides