Kazuki Kitamura

Cell cycle dependent gene expressions and activities of protein phosphatases PP1 and PP2A in mouse NIH3T3 fibroblasts

We determined the mRNA levels and the activities in nuclear and non-nuclear fractions of protein phosphatase type 1 (PP1) and type 2A (PP2A) through the cell cycle in synchronized mouse NIH3T3 fibroblasts. The mRNA level for PP1 alpha was gradually elevated in late G1 phase, began to decrease in M phase, and reached the control level with entering into the next G1 phase. The mRNA level for PP2A was rapidly increased in early G1 phase, kept at the high level, and decreased after S phase. In nuclear fractions of cells, spontaneous activities of both PP1 and PP2A were gradually increased until M phase and rapidly decreased with entering the next G1 phase, while in non-nuclear fraction such dramatic alterations were not observed. Potential activities of PP1 in both fractions revealed by Co(2+)-trypsin treatment showed an oscillaion patterns similar to those of the spontaneous activities. These results strongly suggest that cell cycle dependent gene expressions and activities of PP1 and PP2A play roles in DNA synthesis and mitosis during the cell cycle.

Gene expressions and activities of protein phosphatases PP1 and PP2A in rat liver regeneration after partial hepatectomy

We have examined the levels of gene expressions and activities of protein phosphatases, PP1 and PP2A, in rat regenerating livers. PP1 alpha mRNA started to increase from 6 h after partial hepatectomy (PH) and showed two peaks at 12 and 48 h. PP2A mRNA level showed two peaks at 6 and 10-12 h. Protein phosphatase activities were determined both in non-nuclear fraction and in nuclei. While spontaneous PP1 activity in non-nuclear fraction was nearly constant, potential PP1 activity revealed by Co(2+)-trypsin treatment showed a small peak between 7 and 12 h. In nuclei, both spontaneous and potential PP1 activity began to increase from 4-7 h after PH, reached a maximum (about 2.5-fold over control levels) at 12 h, the time which corresponds to the G1 to S transition in the cell cycle, and then declined back to control levels by 7 days. PP2A activity in non-nuclear fraction was nearly constant in both spontaneous and potential forms. PP2A activity in both forms in nuclei was very low throughout. These results suggest the possibility that PP1 in nuclei plays some role in the G1 to S transition in the cell cycle of hepatocyte proliferation.

Molecular cloning and analysis of the 5′-flanking region of the rat PP1 α gene

Abstract We have cloned an 8 kbp genomic fragment of 5′-flanking region of the gene encoding the catalytic subunit of rat protein phosphatase 1α. Neither CAAT box nor TATA box was detected but a 300 bp high GC region containing nine Sp1 transcription factor binding sites is present immediately upstream of the translation start site, demonstrating that PP1α is a housekeeping gene. Luciferase reporter assay showed that transcription of PP1α is controlled at the high GC region.