B56-Associated Protein Phosphatase 2A Is Required For Survival and Protects from Apoptosis in Drosophila melanogaster

Abstract

Protein phosphorylation and specific protein kinases can initiate signal transduction pathways leading to programmed cell death. The specific protein phosphatases regulating apoptosis have been more elusive. Using double-stranded RNA-mediated interference (RNAi), the role of protein phosphatase 2A (PP2A) in cellular signaling was investigated. Knockdown of A or C subunits individually or of combined B subunits led to concurrent loss of nontargeted PP2A subunits, suggesting that PP2A is an obligate heterotrimer in vivo. Global knockdown of PP2A activity or specific loss of redundant B56 regulatory subunits caused cell death with the morphological and biochemical changes characteristic of apoptosis in cultured S2 cells. B56:PP2A-regulated apoptosis required caspases and the upstream regulators dark, reaper, head involution defective, and dp53. In Drosophila embryos, knockdown of B56-regulated PP2A activity resulted in apoptosis and failure of gastrulation, an effect that was blocked by concurrent RNAi of the caspase Drice. B56-regulated PP2A activity appears to be required upstream of dp53 to maintain a critical proapoptotic substrate in a dephosphorylated, inactive state, thereby preventing apoptosis in Drosophila S2 cells.

PP2A holoenzyme assembly: in cauda venenum (the sting is in the tail)

“…The same study also confirmed earlier reports demonstrating that, in yeast, subunit stability is not linked to heterotrimer formation [20][21][22][23][24]. However, in Drosophila melanogaster Schneider cells, the absence of all B-type subunits promotes the degradation of the A and C subunits, and vice versa [25,26], which indicates that PP2A enzymes are obligate trimers in this system. In mammalian cells, catalytic-, PR55/B-and PR61/B 0 subunits are only stable when bound to the A subunit, whereas free [32].…”

Functional analysis of the PP2A subfamily of protein phosphatases in regulating Drosophila S6 kinase

“…4B, inset). As reported previously [30,31] knockdown of the PP2A catalytic subunit also caused a loss of the B56-1 subunit due to instability of the free protein. Depletion of the B56-1 protein had no detectable effect on the basal level of dS6K or the dephosphorylation caused by amino acid starvation (Fig.…”

“…Previous studies showed that the two Drosophila B56 subunits cooperate to promote cell survival [30][31][32]. B56-1 and -2 could also cooperate to regulate the phosphorylation of dS6K.…”

“…Knockdown of the PP2A catalytic subunit causes apoptosis in Drosophila S2 cells [30,31]. In order to determine whether the induction of apoptosis contributed to effects of catalytic subunit knockdown on dS6K phosphorylation, the experiments were also carried out in the presence of the caspase inhibitor Z-VAD-fmk.…”

“…Due to decreased complexity relative to mammalian cells, Drosophila has been a valuable system to study functions of individual catalytic and regulatory subunits within the PP2A subfamily [30][31][32]. The Drosophila genome contains homologs of the PP2A catalytic, scaffold, and regulatory subunits [30,31], as well as homologs of PP4 [33], PP6 [34], and Tap42 [18].…”