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.
Highlights
POSH3 (Plenty of SH3 domains) is a recently discovered proapoptotic protein that appears to be widely expressed in multiple cell types, at low levels
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
We examined the effects of POSH overexpression in an additional cell type, MDA-MB-231 human breast cancer cells
Summary
Cell Culture—HEK293T cells were grown in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 g/ml streptomycin. Human Akt cDNA was cloned by RT-PCR using cDNA prepared from HEK293 cells as described above and the primers myr-Akt2-HA F primer (ATC GAT AAG CTT ATG GGT TCT TCT AAA TCT AAA CCT AAA AAT GAG GTG TCT GTC ATC AAA GAA GGC) and myr-Akt2-HA-R (GAT ATC ACT AGT TCA GGC ATA GTC GGG CAC GTC ATA GGG ATA CTC GCG GAT GCT GGC CGA) The product of this reaction encodes a protein with a myristoylation sequence at the N terminus and a hemagglutinin epitope tag at the C terminus and was subcloned into the vector pCDNA3.1(ϩ), using engineered HindIII and EcoRV sites. 1 mg of lysate protein was incubated with glutathione-Sepharose beads in binding buffer (50 mM NaCl, 50 mM NaF, 10 mM Tris, pH 7.4, 5 mM EDTA, 1 mM Na3VO4) overnight at 4 °C with rocking to recover GST-POSH.
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