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A full-length complementary DNA (cDNA) clone (pTK-3) encoding an isoform of Mg2+-dependent protein phosphatase β (MPPβ-4) was isolated for the first time from a mouse melanocyte cDNA library. It was strongly suggested that the mRNA corresponding to the pTK-3 insert was a splicing variant of a single pre-mRNA that also encodes MPPβ-1 and -2 (T. Terasawa, T. Kobayashi, T. Murakami, M. Ohnishi, S. Kato, O. Tanaka, H. Kondo, H. Yamamoto, T. Takeuchi, and S. Tamura, 1993, Arch. Biochem. Biophys. 307, 342-349). The amino acid sequence of MPPβ-4 differed from those of MPPβ-1 and -2 only at the carboxyl terminal region. Analysis by reverse transcriptase polymerase chain reaction (RT-PCR) revealed that MPPβ-4 mRNA was expressed only in testis and intestine and not in other mouse tissues tested. Specific expression of the mRNA signals of two other isoforms of MPPβ, MPPβ-3 and -5 (a novel isoform), in testis and intestine was also demonstrated by the RT-PCR. The carboxyl terminal region of MPPβ-5 was found to have a chimera structure composed of part of MPPβ-1 and part of MPPβ-3. The recombinant MPPβ-3 and -4 and the putative MPPβ-5 expressed in Escherichia coli cells exhibited Mg2+-dependent and okadaic acid-insensitive protein phosphatase activities. It was demonstrated that the mRNA expression levels of MPPβ-3, -4, and -5 alter according to the maturation of mouse testis. These resuits suggest that the complex structure of MPPβ isoforms and their tissue- and developmental stage-specific expression reflect the variety of their physiological functions.Copyright 1995, 1999 Academic Press, Inc.

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.