The alpha4-containing form of protein phosphatase 2A in liver and hepatic cells.

The Ser/Thr phosphatase PP2A is a set of multisubunit enzymes that regulate many cellular processes. In yeast, the PP2A regulatory subunit Tap42 forms part of the target of rapamycin (TOR) signaling pathway that links nutrient and energy availability to cell growth. The physiological intersection between the mammalian orthologs of Tap42 and TOR, alpha4 and mTOR, has not been fully characterized. We used two in vivo models of liver growth in the rat, late gestation fetal development and regeneration after partial hepatectomy, to explore the regulation of the alpha4-containing form of PP2A. The alpha4/PP2A catalytic subunit (alpha4/PP2A-C) complex was present in both fetal and adult liver extracts. There was a trend towards higher levels of alpha4 protein in fetal liver, but the complex was more abundant in adult liver. Fractionation of extracts by ion exchange chromatography and transient transfection of the AML12 mouse hepatic cell line indicated that alpha4 associates with PP2A-C but that these complexes have low catalytic activity with both peptide and protein substrates. alpha4 was able to associate with forms of PP2A-C that were both methylated and non-methylated at the carboxy-terminus. The mTOR inhibitor rapamycin did not block the formation of alpha4/PP2A-C in liver or hepatic cells, nor did it appear to modulate PP2A activity. Furthermore, sensitivity to the growth inhibitory effects of rapamycin among a panel of hepatic cell lines did not correlate with levels of alpha4 or alpha4/PP2A-C. Our results indicate that the yeast Tap42/TOR paradigm is not conserved in hepatic cells.

Subunit composition and developmental regulation of hepatic protein phosphatase 2A (PP2A).

The prototypical form of the Ser/Thr phosphatase PP2A is a heterotrimeric complex consisting of catalytic subunit (C), and A and B regulatory subunits. C-terminal methylation of PP2A-C influences holoenzyme assembly. Using late gestation development in the rat as an in vivo model of liver growth, we found that PP2A-C protein and activity levels were higher in fetal compared to adult liver extracts. However, unmethylated PP2A-C was much higher in the adult extracts. In MonoQ fractionation, unmethylated C eluted separately from methylated C, which was present predominantly in ABC heterotrimers. Gel filtration chromatography revealed that some unmethylated C was present as free catalytic subunit in adult liver. In addition, a significant proportion of PP2A was in inactive forms that may involve novel regulatory subunits. Our results indicate that methylation of PP2A-C appears to be a primary determinant for the biogenesis of PP2A heterotrimers.