Although members of the PP2A subfamily of serine/threonine phosphatases have been implicated in the TOR signaling pathway, the identity of the phosphatase that dephosphorylates the TOR phosphorylation site on S6K has not been determined. This study supports the conclusion that this site is dephosphorylated by PP2A, and not by other members of the PP2A subfamily or other classes of protein phosphatase. The ability of a PP2A-selective concentration of calyculin A to nearly completely block dephosphorylation of dS6K argues that only the PP2A subfamily is involved in dephosphorylating dS6K. The knockdown experiments revealed that PP2A is the only member of this subfamily that is likely to play a direct role in dephosphorylation of Thr398. A direct role for PP2A in dephosphorylating S6K is consistent with data showing that the catalytic subunit of PP2A can be isolated in complexes with mammalian S6K [20
] and previous experiments showing that knockdown of PP2A in Drosophila
S2 leads to enhanced basal dS6K phosphorylation [18
]. While the data are consistent with a direct role of PP2A in dephosphorylation of Thr398, the effect of PP2A dsRNA could also be due to altered phosphorylation of another component of the dTOR pathway.
While the loss of PP2A substantially increased S6K phosphorylation levels, RNAi-mediated depletion had a partial affect. The increase in basal dS6K phosphorylation was moderate compared to calyculin A treatment and the inhibition of dephosphorylation induced by removal of amino acids was not complete. The residual dephosphorylation was most likely due to incomplete knockdown of the PP2A catalytic subunit. Under basal conditions (complete medium), dTOR is active and a partial decrease in phosphatase activity would result in elevated steady-state levels of dS6K phosphorylation. Following removal of amino acids, TOR activity would be decreased leading to a new steady level of dS6K phosphorylation that would also be elevated in cells depleted of PP2A. The suggestion that dTOR remains at least partially active following removal of amino acids is supported by observations that amino acids only weakly activate S6K in Drosophila
]. In contrast, when dTOR activity in the TORC1 complex is strongly inhibited by rapamycin, the residual PP2A could still lead to a complete, albeit slower, dephosphorylation of S6K. While the data presented here show that PP2A plays a dominant role in dephosphorylation of the TOR site on S6K, they cannot rule out a possible contribution from another calyculin A-sensitive phosphatase.
Depletion of dPP4 by RNAi led to a 20% reduction in basal phosphorylation of dS6K and a greater extent of dephosphorylation following amino acid starvation. While they indicate it is unlikely that dPP4 plays a direct role in dephosphorylation of dS6K, these results suggest a role for dPP4 in the dTOR pathway upstream of dS6K. The decrease in dS6K phosphorylation may contribute to the 20% decrease in cell growth previously observed in S2 cells depleted of dPP4 [31
]. PP4 has been implicated in a variety of regulatory processes [24
], but has not been extensively studied in Drosophila
. Reduction in dPP4 levels results in a semi-lethal phenotype in Drosophila
early embryos and defects in microtubule assembly [44
]. A decrease in dTOR signaling might also contribute to reduced viability caused by deficiency of dPP4.
Based on the currently accepted model for regulation of PP2A [26
], it was expected that the catalytic subunit or core dimer would be targeted to dS6K by a known PP2A regulatory protein. Consequently, knockdown of the targeting protein should inhibit dephosphorylation of Thr398 by preventing interaction of catalytic subunit and dS6K. The Drosophila
genome contains homologs of each of the characterized families of PP2A regulatory subunits [30
]. Knockdown of any of the four Drosophila
PP2A regulatory subunits did not affect the basal level of dS6K phosphorylation of Thr398 nor decrease the rate or extent of dephosphorylation induced by amino acid starvation. These results suggested that dephosphorylation of Thr398 by PP2A is not mediated by one of these proteins. Although the knockdown of each of the regulatory subunits was highly efficient, the absence of an effect on dS6K phosphorylation could be due to incomplete knockdown. Alternatively, targeting to dS6K could be mediated by a novel PP2A subunit or another PP2A interacting protein. Mammalian cells contain a two related proteins, striatin and SG2NA, that also interact with the PP2A core dimer [45
]. These proteins have been linked to regulation of the cytoskeleton [46
], lipid raft formation [47
], and non-genomic actions of the estrogen receptor [48
]. BLAST searches of the Drosophila
genome identified a gene (Cka
) encoding a transcript with 36% identity to human striatin and SG2NA. The Drosophila
CKA protein regulates signaling through the dJNK protein kinase pathway [49
]. Although there is no evidence that striatin, SG2NA, or CKA are involved in the TOR pathway, it is possible that CKA targets Drosophila
PP2A to dS6K. The inability of regulatory subunit knockdown to increase S6K phosphorylation could also be due to a pool of free AC core dimer that mediates dephosphorylation of Thr398.
Depletion of the Drosophila
B56-2 regulatory subunit caused a significant increase in the rate and extent of dS6K dephosphorylation induced by amino acid starvation. The data suggest that the dB56-2/PP2A complex may act upstream of dS6K to promote dTOR activity. The Drosophila
B56-2 gene (widerborst/wdb
) is essential for cell viability as complete loss of WDB function is lethal. Genetic analysis has demonstrated that WDB plays roles in regulating tissue polarity during development [50
] and regulation of the Drosophila
circadian clock [51
]. While the results presented here indicate that dB56-2 is not directly involved in regulating dS6K, the decrease in phospho-Thr398 levels in S2 cells depleted of the protein may reflect an additional role for dB56-2 in the regulation of dTOR activity.
Based on the homology between the yeast, Drosophila
, and mammalian Tap42/α4 proteins, another candidate for targeting PP2A to dS6K was the dTap42 protein. However, knockdown of dTap42 had no detectable affect on basal phosphorylation of Thr398 or on the rate or extent of dephosphorylation caused by amino acid starvation or rapamycin treatment. In addition, co-knockdown of dTap42 had no major effect on the enhanced dS6K phosphorylation caused by knockdown of the PP2A catalytic subunit. These results are consistent with a previous study showing knockdown of dTap42 had no effect on basal phosphorylation of dS6K [18
]. This study also showed that disruption of the dTap42
gene had no affect on dTOR-mediated cell growth. The lack of effect on Thr398 phosphorylation in cells depleted of dTap42 was inconsistent with a role of this protein in directing PP2A to S6K. It has been reported that α4/mTap42, S6K, and PP2A interact in stimulated splenic B cells [17
]. The data presented here suggest that such a complex is not present in Drosophila
cells or that it mediates dephosphorylation of sites on dS6K other than the TOR site at Thr398. As observed previously in Drosophila
imaginal disks [18
] and for the α4/mTap42 protein in murine thymocytes [52
], loss of dTap42 resulted in apoptosis of S2 cells (not shown). These observations are all consistent with an essential role for the Tap42 homologs in cell survival. Since the PP2A catalytic subunit is also essential for cell survival [30
], it is possible that some of the pro-survival actions of this phosphatase are mediated by Tap42.