Okadaic Acid Suppresses Neural Differentiation-dependent Expression of the Neurofilament-L Gene in P19 Embryonal Carcinoma Cells by Post-transcriptional Modification

Abstract

Mouse P19 embryonal carcinoma cells in aggregation culture in the presence of 10(-6) M retinoic acid followed by monolayer culture differentiate into nerve and glial cells. In this study, we demonstrated that the neurofilament-L (NF-L) mRNA and protein levels of these cells were enhanced in accordance with their retinoic acid-induced neural differentiation. Okadaic acid (OA) treatment of the cells markedly suppressed this differentiation-dependent NF-L gene expression increase and neurite outgrowth of the cells. Similar results were obtained when tautomycin was used instead of OA, suggesting that inhibition of protein phosphatase(s) is involved in the suppression of neural differentiation. OA treatment did not affect the NF-L gene transcription level, determined by the nuclear run-on transcription assay, but it did reduce the stability of both the 3.5- and 2.3-kilobase NF-L mRNAs. The expression and activity levels of protein phosphatase 2A (PP2A) and 2B (PP2B) but not protein phosphatase 1 (PP1) in P19 cells increased in accordance with the enhanced NF-L gene expression. The presence of OA in the culture medium during the course of the neural differentiation caused a reduced PP2A activity but not PP1 and PP2B activities of the cell extracts. On the other hand, both PP1 and PP2B activities but not PP2A activity of cell extracts were suppressed by the addition of cyclosporin A or FK506 in the culture medium. However, both cyclosporin A and FK506 treatments affected neither NF-L gene expression nor neurite outgrowth. These results demonstrate that the OA treatment inhibits the differentiation-dependent increase in NF-L gene expression by destabilizing its mRNAs and suggest that PP2A plays key roles in the differentiation-dependent enhanced expression of the NF-L gene and is the point of the action of OA.

Vertebrate endothelial lipase: comparative studies of an ancient gene and protein in vertebrate evolution

“…The three LIPP and LIPG domains were readily apparent, including the N-terminal ‘lipase’ domain with the active site triad residues buried under the ‘lid’ domain observed for horse LIPP. The ‘lid’ has been previously shown to contribute to the preference for triglyceride and phopholipid substrates of other vascular lipases (LIPC and LIPL) (Dugi et al, 1995; Kobayashi et al, 1996) and may play a major role in determining the preference for phospholipid substrates for LIPG. A ‘hinge’ region was also observed for these vertebrate LIPG proteins, separating the ‘lipase’ and ‘plat’ domains, with the latter having a ‘sandwich-like’ β-pleated sheet structure.…”

“…Predicted secondary structures and tertiary structures for vertebrate LIPG proteins showed a strong similarity with human and horse LIPP (Winkler et al, 1990; Bourne et al, 1994). Three major structural domains were apparent for vertebrate LIPG, including the ‘lipase’ domain containing the catalytic triad residues; the ‘lid’ which covers the active site and may contribute to the substrate specificities of neutral lipases (Dugi et al, 1995; Kobayashi et al, 1996); and the ‘plat’ domain which contributes to lipoprotein binding (Wong et al, 1991). Phylogenetic studies using 16 distinct human and mouse lipase sequences indicated that the LIPG gene has appeared early in vertebrate evolution, prior to the appearance of bony fish more that 500 million years ago.…”

Comparative studies of vertebrate lipoprotein lipase: A key enzyme of very low density lipoprotein metabolism

“…The three LPL and LIPP domains were readily apparent, including the N-terminal `lipase' domain with the active site triad residues buried under the `lid' domain observed for horse LIPP. The `lid' has been previously shown to contribute to the preference for triglyceride and phospholipid substrates of other vascular lipases (HL and EL) (Dugi et al, 1995; Kobayashi et al, 1996) and may play a major role in determining the preference for triglyceride rich lipoprotein LPL substrates. A `hinge' region was also observed for vertebrate LPL proteins, separating the `lipase' and `plat' domains, with the latter having a `sandwich-like' β-pleated sheet structure.…”

Vertebrate hepatic lipase genes and proteins: a review supported by bioinformatic studies

“…Site-directed mutagenesis studies of site 3 (human HL 78Asn) have demonstrated that this N-glycosylation site is required for the efficient secretion of this liver enzyme. 64,65 …”

“…Predicted secondary structures and tertiary structures for vertebrate HL proteins showed a strong similarity with human and horse pancreatic lipases (LIPP). 29,30 Three major structural domains were apparent for vertebrate HL, including the “lipase” domain containing the catalytic triad residues; the “lid”, which covers the active site and may contribute to the substrate specificities of neutral lipases; 31,64 and the “plat” domain, which contributes to lipoprotein binding. 65 Phylogenetic studies using amino acid sequences for 13 vertebrate HL lipases, human and mouse LPL and EL, and an invertebrate lipase indicated that the LIPC gene has appeared early in vertebrate evolution, probably prior to the appearance of bony fish more that 500 million years ago.…”