Identification of okadaic acid-induced phosphorylation events by a mass spectrometry approach.
Okadaic acid (OA) is a widely used small-molecule phosphatase inhibitor that is thought to selectively inhibit protein phosphatase 2A (PP2A). Multiple studies have demonstrated that PP2A activity is compromised in the brains of Alzheimer's disease patients. Thus, we set out to determine changes in phosphorylation that occur upon OA treatment of neuronal cells. Utilizing isotope-coded affinity tags and mass spectrometry analysis, we determined the relative abundance of proteins in a phosphoprotein enriched fraction from control and OA-treated primary cortical neurons. We identified many proteins whose phosphorylation state is regulated by OA, including glycogen synthase kinase 3beta, collapsin-response mediator proteins (DRP-2, DPYSL-5, and CRMP-4), and the B subunit of PP2A itself. Most interestingly, we have found that complexin 2, an important regulator of neurotransmitter release and synaptic plasticity, is phosphorylated at serine 93 upon OA treatment of neurons. This is the first report of a phosphorylation site on complexin 2.
Hill
Jennifer J
JJ
Institute for Biological Sciences, National Research Council Canada, 100 Sussex Dr., Ottawa, Ont., Canada K1A 0R6. Jennifer.Hill@nrc.gc.ca
Callaghan
Deborah A
DA
Ding
Wen
W
Kelly
John F
JF
Chakravarthy
Balu R
BR
D000595:Amino Acid Sequence; D000818:Animals; D002478:Cells, Cultured; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000032:analysis; D019319:Okadaic Acid / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D010750:Phosphoproteins / Q000032:analysis* / Q000737:chemistry; D010766:Phosphorylation / Q000187:drug effects; D054648:Protein Phosphatase 2; D051381:Rats; D017207:Rats, Sprague-Dawley; D015203:Reproducibility of Results; D012107:Research Design; D016875:tau Proteins / Q000032:analysis
D009419:Nerve Tissue Proteins; D010750:Phosphoproteins; D016875:tau Proteins; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2