Regulation of the Pro-apoptotic Scaffolding Protein POSH by Akt

Abstract

POSH (Plenty of SH3 domains) binds to activated Rac and promotes apoptosis by acting as a scaffold to assemble a signal transduction pathway leading from Rac to JNK activation. Overexpression of POSH induces apoptosis in a variety of cell types, but apoptosis can be prevented by co-expressing the pro-survival protein kinase Akt. We report here that POSH is a direct substrate for phosphorylation by Akt in vivo and in vitro, and we identify a major site of Akt phosphorylation as serine 304 of POSH, which lies within the Rac-binding domain. We further show that phosphorylation of POSH results in a decreased ability to bind activated Rac, as does phosphomimetic S304D and S304E mutation of POSH. S304D mutant POSH also shows a strongly reduced ability to induce apoptosis. These findings identify a novel mechanism by which Akt promotes cell survival. POSH3 (Plenty of SH3 domains) is a recently discovered proapoptotic protein that appears to be widely expressed in multiple cell types, although at low levels. POSH was first identified as a binding partner of activated Rac and has been shown to act as a scaffolding protein in a kinase cascade signaling pathway that leads to apoptotic cell death (1, 2). In this pathway, Rac activates one of the mixed lineage kinases (MLKs, a group of MAPKKKs), which in turn phosphorylate and activate MKK4 and/or MKK7 (which are MAPKKs) which then phosphorylate and activate c-Jun N-terminal kinases (JNKs, one group of MAPKs) (2). Activated JNKs induce release of cytochrome c from mitochondria and trigger subsequent apoptosis. POSH directly binds Rac, MLK, and another scaffold protein, JIP (JNK-interacting protein), which in turn binds MKK4/7 and JNK, to facilitate this pathway. This multiprotein signaling assembly has been termed PJAC, for POSH-JIP apoptotic complex (3). The role of POSH as a scaffold for this signaling complex appears to be critical; in an apoptotic model involving withdrawal of nerve growth factor from cultures of neuronally differentiated PC12 cells or rat primary sympathetic neurons, apoptosis was dramatically reduced by pretreatment with POSH short interfering RNA or antisense oligonucleotides (4).The decision of a cell to undergo apoptosis is not undertaken lightly; apoptotic pathways are subject to regulation at many levels, and cells must integrate a variety of pro-apoptotic and anti-apoptotic signals. Regulation of the PJAC apoptotic pathway appears to follow this pattern, with multiple regulatory interactions. One form of regulation of PJAC may lie in the expression level of POSH protein within cells. POSH is maintained within healthy cells at very low levels, at least in part by POSH auto-ubiquitination and proteasomal degradation (5). Increasing the level of POSH protein by microinjection or ectopic expression induces apoptosis in a variety of cell types (1, 2, 4, 6 -9).Another potential regulator of the POSH-JIP apoptotic complex appears to be the pro-survival kinase Akt, also known as protein kinase B. Three closely related Akt genes exist (AKT1-3) that have b...

PI3K/Akt: getting it right matters

“…These authors showed that the binding of Akt2 to POSH results in an inhibition of JNK activity, and that this inhibition is mediated by phosphorylation of the upstream kinase MLK3 and leads to the disassembly of the JNK signaling complex. In turn, POSH is also an Akt substrate (Lyons et al, 2007). Taken together, these findings point to an intriguing model for the regulation of the JNK pathway by Akt, in which the Aktdependent phosphorylation of specific components can block signal transduction through the stress-regulated kinase cascade.…”

The Scaffold Protein POSH Regulates Axon Outgrowth

“…Membrane trafficking, apoptosis, calcium homeostasis, and process outgrowth inhibition each require the RING domain of POSH, suggesting a conserved function for this domain (Alroy et al, 2005;Tsuda et al, 2005;Kim et al, 2006;Tuvia et al, 2007). POSH interacts with JNK pathway signaling components (Tapon et al, 1998;Xu et al, 2003), the AKT serine threonine kinase Lyons et al, 2007), and the small GTPase Rac (Tapon et al, 1998). All are known regulators of cytoskeletal dynamics; however, their contribution to POSH-mediated process outgrowth regulation remains to be determined.…”

“…POSH interacts with activated Rac, which activates a subset of MLK family members, notably the Cdc42/Rac-interactive binding domain containing MLK1-3 (Tapon et al, 1998;Gallo and Johnson, 2002). Akt negatively regulates POSH-associated JNK signaling and Rac binding Lyons et al, 2007). POSH functions as a proapoptotic protein in fibroblasts, mature neurons, and Xenopus embryos by activating JNK signaling (Tapon et al, 1998;Xu et al, 2003Xu et al, , 2005Kim et al, 2005;Zhang et al, 2006).…”

Protein kinase B: signalling roles and therapeutic targeting

“…Additionally, the scaffold protein plenty of SH3 domains (POSH) has recently been identified as a substrate for PKB. This phosphorylation decreases binding of POSH to Rac, again leading to lowered JNK activity [99]. Both carboxyl-terminal regulator protein (CTMP) and APE bind to the C terminus of PKB and have been reported to enhance PKB phosphorylation and activation [100,101].…”