The α4‐containing form of protein phosphatase 2A in liver and hepatic cells

PP2A is a major Ser/Thr phosphatase that plays a significant role in the regulation of many cellular processes, including metabolism, transcription, translation, cell cycle progression, cell growth and apoptosis [Janssens and Goris, 2001; Mumby and Walter, 1993; Sontag, 2001; Zolnierowicz, 2000]. The activity of such a versatile enzyme must therefore be tightly controlled in vivo. Indeed, the catalytic subunit of PP2A (PP2A-C) is regulated at many levels. The C-terminus of PP2A-C is post-translationally modified by phosphorylation at Y307 and methyl esterification at L309. Phosphorylation results in inhibition of PP2A activity [Chen et al., 1992; Guo and Damuni, 1993] while methylation is thought to play an important function in regulatory subunit binding [Bryant et al., 1999; Tolstykh et al., 2000; Wu et al., 2000; Yu et al., 2001]. More specifically, PP2A-C methylation can influence the association of PP2A-C with specific regulatory subunits, thereby serving to determine the composition, subcellular localization and function of PP2A-C holoenzymes in a variety of cell types [Gentry et al., 2005; Longin et al., 2007; Nunbhakdi-Craig et al., 2007]. The prototypical PP2A enzyme is a heterodimer that consists of the catalytic C subunit and scaffolding A subunit. The substrate specificity and subcellular localization of PP2A are thought to be defined by an array of B-type regulatory subunits that bind to the AC dimer to form a variety of heterotrimeric complexes [Mumby and Walter, 1993]. Among various B-type subunits, B56α/β/e complexed with the A and C subunits localize to the cytoplasm, whereas heterotrimeric forms of PP2A that include B56δ/γ1/γ3 are concentrated in the nucleus [McCright et al., 1996]. Likewise, the two regulatory subunits Cdc55p (a B-type subunit) and Rts1p (a B′-type subunit) of Saccharomyces cerevisiae display distinct, dynamic localization patterns that support the hypothesis that regulatory subunits determine localization of PP2A [Gentry and Hallberg, 2002]. Recent structural studies further support the role of the B subunit in determining substrate specificity by showing that regulatory B′ subunit interacts with the PP2A-C subunit near the active site and that the B′ subunit is responsible for the ultimate tertiary structure of the catalytic subunit [Cho and Xu, 2007; Xu et al., 2006]. Studies in yeast have established a model in which PP2A, through its interaction with Tap42, functions as a component of the rapamycin-sensitive TOR signaling pathway, a pathway required for initiation of protein synthesis in response to nutrient availability. In this model, TOR phosphorylates Tap42 to promote association with the catalytic subunits of several Ser/Thr phosphatases, including the yeast homologs of PP2A-C [Jiang Y and Broach JR, 1999]. Under nutrient-rich conditions, phosphorylated Tap42 binds to and inhibits PP2A activity towards downstream effectors of TOR, such as the transcriptional regulator GLN3 [Beck and Hall, 1999] and the Ser/Thr kinase NPR1 [Schmidt et al., 1998]. In contrast, nutrient starvation or rapamycin block the phosphorylation of Tap42, inducing the dissociation of Tap42 from PP2A-C [Di Como CJ and Arndt KT, 1996]. Thus, the overall model suggests that TOR regulates protein phosphorylation in yeast by indirectly restraining the activity of PP2A through phosphorylation of Tap42. In mammalian cells, PP2A-C has been found to associate with the homolog of Tap42, a novel regulatory protein termed α4 [Murata et al., 1997; Prickett and Brautigan, 2004]. α4 binds to PP2A-C independent of the A and B subunits. Thus, α4/PP2A-C is considered to represent a unique, heterodimeric form of the phosphatase [Jiang Y and Broach JR, 1999]. Whether mTOR regulation of α4/PP2A-C plays a role analogous to that in yeast is controversial. Some studies suggest that mTOR can directly phosphorylate its effectors, such as S6K1 and 4E-BP1, while others claim that the mTOR-dependent phosphorylation of S6K1 and 4E-BP1 is an indirect result of the inhibition of PP2A [Gingras AC et al., 2001a; Gingras AC et al., 2001b]. PP2A is able to dephosphorylate S6K1 in vitro [Ferrari et al., 1991], and okadaic acid, a potent PP2A inhibitor, antagonizes dephosphorylation of TOR substrates (i.e. S6K1 and 4E-BP1), promoting S6K1 activation and cell growth in Drosophila [Cygnar et al., 2005]. A role for PP2A is supported by studies showing a direct interaction of PP2A with S6K1 in mammalian cells [Peterson RT et al., 1999; Westphal et al., 1999]. The α4 protein has been found to associate with the catalytic subunits of PP2A, PP4 and PP6, making it a common regulator of multiple PP2A family members [Chen et al., 2004; Kloeker et al., 2003; Murata et al., 1997]. Mutations in PP2A-C that induce the loss of binding to A subunit enhance its binding to α4, whereas mutations that eliminate the α4 association increase AC content [Prickett and Brautigan, 2004]. This suggests a competitive and mutually exclusive association of A subunit and α4 with PP2A-C in cells. Furthermore, previous studies show that single mutations of PP2A-C at the site of phosphorylation (Y307F) or methyl esterification (L309Q) result in recovery of only AC dimers whereas double mutation of both residues favor the formation of α4/PP2A-C [Chung et al., 1999]. This suggests a role for PP2A-C post-translation modification in the association with α4. Recent structural studies reveal that the tetratricopeptide repeat-like fold in Tap42/α4 may have a scaffolding function that promotes the dephosphorylation of substrates recruited to the C-terminal region of PP2A-C [Yang et al., 2007]. It has been proposed that α4, rather than simply inhibiting PP2A activity, may protect mTOR downstream effectors from being dephosphorylated by modulating the activity of other undefined phosphatases [Inui et al., 1998]. It has been shown that α4 interacts with S6K1 through PP2A-C in a rapamycin-sensitive manner [Yamashita et al., 2005]. Recent studies in Drosophila have suggested that PP2A, but not other members of this subfamily, is the major S6K phosphatase [Bielinski and Mumby, 2007]. Studies in Drosophila led to the interesting observation that Tap42 induces nuclear accumulation of PP2A-C but loss of TOR has no effect on PP2A-C localization [Cygnar et al., 2005]. This suggests that Tap42 may act independently of TOR and regulate the localization of PP2A, similar to the B regulatory subunits. A number of questions regarding the function and regulation of the mTOR/α4/PP2A system remain unanswered. Rapamycin sensitivity of the interaction between α4 and PP2A-C has been observed in some systems [Inui et al., 1998; Kong et al., 2004; Murata et al., 1997], but in others this association is unaffected by rapamycin [Kloeker et al., 2003; Nanahoshi et al., 1998; Nien et al., 2007]. In addition, the issue of whether α4 has a positive or negative effect on PP2A activity has not been definitively determined. Early studies indicated that yeast strains that overexpressed Tap42 mutants were almost completely resistant to rapamycin [Di Como CJ and Arndt KT, 1996]. α4 transfection in Jurkat cells was shown to result in rapamycin resistance [Inui et al., 1998]. Conditions that promote the association of Tap42 with the yeast homologs of PP2A-C, such as deletion of TPD3 (yeast A subunit) or CDC55 (yeast B subunit), induce partial rapamycin resistance [Jiang Y and Broach JR, 1999]. These relationships between a4 and resistance to the anti-proliferative effects of rapamycin have been relatively unexplored in mammalian models for rapamycin resistance. In the present studies, we have explored the in vivo relationship between mTOR, α4 and PP2A-C in liver. We used two in vivo models of liver growth, the rapamycin-resistant hepatocyte proliferation that occurs in the late gestation fetal rat, and the rapamycin-sensitive process of liver regeneration induced by 2/3 partial hepatectomy in the adult rat [Curran et al., 1993; Fausto and Webber, 1993; Gruppuso et al., 1997]. We also utilized in vitro hepatic cell models in an effort to facilitate the study of the functional regulation of PP2A. The normal diploid nontumorigenic rat hepatic epithelial line (WB-F344, abbreviated as WB) was originally isolated from an adult Fischer rat [Tsao MS et al., 1984]. We have utilized a spectrum of cell lines derived from the WB line or from other sources [Campbell et al., 2000; Hixson et al., 1985] that we have found exhibit varying degrees of rapamycin sensitivity. Based on the limited transfection efficiency seen with these rat hepatic cell lines, we also utilized a hepatic cell line derived from mice transgenic for human TGFα, AML12 [Wu et al., 1994]. Overall, our studies have found that the regulation of the association between PP2A-C and α4 and the effect of rapamycin on the interaction in hepatic cells do not follow the yeast paradigm.