| Research Abstract |
Paired helical filaments (PHF) are closely related to neuronal death and thus their complete elucidation should lead us to understanding of particular intracellular environmental alterations that cause neurons to die. I focused on the abnormal phosphorylation of PHF-tau because the phosphorylation could precede the PHF formation.
Two phosphorylation-dependent PHF polyclonals and two PHF monoclonals each of which has distinct specificity has been shown to recognize fetal tau but not adult tau. This observation led us to determine the phosphorylation sites in fetal tau as a first step. Fetal tau bears at most ten covalently bound phosphates, eight of which are proline-directed. The phosphorylation sites are roughly equal to those phosphorylated by cdk5 and GSK3beta. Two non proline-directed phosphorylated sites are found to be indeed the motifs for GSK3beta, when downstream proline-directed sites are phosphorylated.
We next attempted to determine the phosphorylation sites in PHF in a simil
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ar way. Major difficulaties were unusual resistance to many specific proteases and very poor recovery of phosphopeptides from a HPLC column, which was never experienced in fetal tau. PHF-tau is phosphorylated at most twenty One sites ; Ten are proline-directed, while eleven are non proline-directed. Most of the sites phosphorylated in fetal tau are also phosphorylated in PHF-tau, which explains those immunochemical similarities between PHF-tau and fetal tau. Overall, the phosphorylation of PHF can be considered to consist of fetal-type phosphorylation plus additional proline-directed and non proline-directed phosphorylation. Some of those non proline-directed sites correspond to the sites phosphorylated by cAMP-dependent protein kinase (PKA). Thus, the phosphorylation of PHF-tau would be mediated by multiple kinases, cdk5, GSK3beta, and possibly PKA.Those non proline-directed phosphorylation which distinguish PHF-tau from fetal tau may be important to the unusual characteristics of PHF-tau, including its assembly incompetence and protease resistance. Less
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