Identification and characterization of the novel centrosomal protein centlein.

Centrosomes function as the major microtubule (MT)-organizing center. They are composed of a pair of centrioles which are surrounded by the pericentriolar material. Here, we describe the molecular characterization of a novel protein, named centlein (centrosomal protein). Centlein was a protein of 721 amino acids with a calculated molecular weight of 82,717 and possessed coiled-coil domains. Western blot analysis indicated that centlein was ubiquitously expressed. Endogenous centlein as well as enhanced green fluorescent protein-fused centlein was localized at centrosomes in interphase and mitosis. When centrosomes were isolated from cells treated with nocodazole, an MT-disrupting agent, centlein and the centrosomal protein, gamma-tubulin, were enriched in the same fractions. These data indicate that centlein is a widespread centrosomal protein and that its association with centrosomes is independent of MTs. Centlein appeared to be enriched in the mother centriole in G1 phase, suggesting possible involvement of centlein in mother-centriole-related functions, such as duplication of centrioles and generation of primary cilia.

Involvement of CaM kinase II in gonadotropin-releasing hormone-induced activation of MAP kinase in cultured hypothalamic neurons.

Gonadotropin-releasing hormone (GnRH) is secreted from hypothalamic GnRH neurons. There is accumulating evidence that GnRH neurons have GnRH receptors and that the autocrine action of GnRH activates MAP kinase. In this study, we found that KN93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinases (CaM kinases), inhibited the GnRH-induced activation of MAP kinase in immortalized GnRH neurons (GT1-7 cells). Immunoblot analysis indicated that the CaM kinase IIdelta2 isoform (CaM kinase IIdelta2) and synapsin I were expressed in GT1-7 cells. GnRH treatment rapidly increased phosphorylation of synapsin I at serine 603, a specific phosphorylation site for CaM kinase II, suggesting that GnRH treatment rapidly activated CaM kinase IIdelta2. In addition, when we stably overexpressed CaM kinase IIdelta2 in GT1-7 cells, the activation of MAP kinase was strongly enhanced. These results suggest that CaM kinase IIdelta2 was involved in the GnRH-induced activation of MAP kinase in GT1-7 cells.

Phosphorylation of tau at serine 416 by Ca2+/calmodulin-dependent protein kinase II in neuronal soma in brain.

It is well known that tau is a good in vitro substrate for Ca2+/calmodulin-dependent protein kinase II (CaM kinase II). However, it is not clear at present whether CaM kinase II phosphorylates tau in vivo or not. Serine 416, numbered according to the longest human tau isoform, has been reported to be one of the major phosphorylation sites by CaM kinase II in vitro. In this study, we produced a specific antibody against tau phosphorylated at serine 416 (PS416-tau). Immunoblot analysis revealed that the antibody reacted with tau in the rat brain extract which was prepared in the presence of protein phosphatase inhibitors. Developmental study indicated that serine 416 was strongly phosphorylated at early developmental stages in rat brain. We examined the localization of PS416-tau in primary cultured hippocampal neurons and the immortalized GnRH neurons (GT1-7 cells), which were stably transfected with CaM kinase IIalpha cDNA. Immunostaining of these cells indicated that tau was phosphorylated mainly in neuronal soma. Interestingly, tau in neuronal soma in Alzheimer's disease (AD) brain was strongly immunostained by the antibody. These results suggest that CaM kinase II is involved in the accumulation of tau in neuronal soma in AD brain.