Phosphorylation sites on tau identified by nanoelectrospray mass spectrometry:: Differences in vitro between the mitogen-activated protein kinases ERK2, c-Jun N-terminal kinase and P38, and glycogen synthase kinase-3β

The stress-activated kinases c-Jun N-terminal kinase (JNK) and p38 are members of the mitogen-activated protein (MAP) kinase family and take part in signalling cascades initiated by various forms of stress. Their targets include the microtubule-associated protein tau, which becomes hyperphosphorylated in Alzheimer's disease. It is necessary, as a forerunner for in vivo studies, to identify the protein kinases and phosphatases that are responsible for phosphate turnover at individual sites. Using nanoelectrospray mass spectrometry, we have undertaken an extensive comparison of phosphorylation in vitro by several candidate tau kinases, namely, JNK, p38, ERK2, and glycogen synthase kinase 3 beta (GSK3 beta). Between 10 and 15 sites were identified for each kinase. The three MAP kinases phosphorylated Ser(202) and Thr(205) but not detectably Ser(199), whereas conversely GSK3 beta phosphorylated Ser(199) but not detectably Ser(202) or Thr(205). Phosphorylated Ser(404) was found with all of these kinases except JNK. The MAP kinases may not be strictly proline specific: p38 phosphorylated the nonproline sites Ser(185), Thr(245), Ser(305), and Ser(356), whereas ERK2 was the most strict. All of the sites detected except Thr(245) and Ser(305) are known or suspected phosphorylation sites in paired helical filament-tau extracted from Alzheimer brains. Thus, the three MAP kinases and GSK3 beta are importantly all strong candidates as tau kinases that may be involved in the pathogenic hyperphosphorylation of tau in Alzheimer's disease.