Antisera to paired helical filaments (PHF) were found to contain a significant amount of tau antibodies specific for a phosphorylated form, but only a negligible amount of those specific for a non-phosphorylated form. Also, the phosphorylated tau-specific antibodies, but not the non-phosphorylated tau-specific ones, labeled neurofibrillary tangles isolated in the presence of sodium dodecyl sulfate (SDS) and stained both tangles and senile plaque neuritis in fixed tissue sections in a very similar way to as the whole antiserum did. Taken together, these results strongly suggest that a major antigenic determinant of PHF is phosphorylated tau itself.
Abstract. Hydrolysis of inositol phospholipids by receptor stimulation activates two separate signaling pathways, one leading to the activation of protein kinase C (C kinase) via formation of diacylglycerol. The other is the inositol trisphosphate (IP3)/Ca 2÷ pathway and a major downstream kinase which is activated is Ca2÷/calmodulin-dependent protein kinase II (CaM kinase II). To examine signaling pathways of C kinase and CaM kinase II to the cytoskeletal protein vimentin, we prepared monoclonal antibodies YT33 and MO82 which recognize the phosphorylation state of vimentin by C kinase and by CaM kinase II, respectively. Ectopic expression of constitutively active C kinase or CaM kinase II in primary cultured astrocytes by microinjection of the corresponding expression vectors induced phosphorylation of vimentin at each specific phosphorylation site, followed by reorganization of vimentin filament networks. In contrast, simultaneous activation of C kinase and CaM kinase II by inositol phospholipid hydrolysis with receptor stimulation led to an exclusive phosphorylation of vimentin at the CaM kinase II site, not at the site of C kinase. These results indicate that the intracellular targeting of C kinase and CaM kinase II signalings to vimentin is regulated separately, under physiological conditions. "r~ECEPTOR-mediated hydrolysis of phosphatidylinosi-1 ~" tol 4,5-bisphosphate (PIP2) relays extracellular l ~. signals into the cell and two kinases, protein kinase C (C kinase) and Ca2÷/calmodulin-dependent protein kinase II (CaM kinase II) play a central role in this signaling. Hydrolysis of PIP2 leads to formation of diacylglycerol and activation of inositol trisphosphate (IP3)-induced Ca 2+ signaling (for reviews see Michell, 1992;Berridge, 1993). C kinase and CaM kinase II, activated by diacylglycerol and Ca 2+, respectively, transmit these two signalings to downstream molecules (for reviews see Nishizuka, 1984Nishizuka, , 1986Colbran et al., 1989; Hanson and Schtflman, 1992). From the viewpoint of signaling cascade, one of the most distinctive features of kinases represented by C kinase and CaM ki-
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