The role of the PI 3-kinase cascade in regulation of cell growth is well established [1]. PKB (protein kinase B) is a key downstream effector of the PI 3-kinase pathway and is best known for its antiapoptotic effects [2,3] and the role it plays in initiation of S phase [4]. Here, we show that PKB activity is high in the G2/M phase of the cell cycle in epithelial cells. Inhibition of the PI 3-kinase pathway in MDCK cells induces apoptosis at the G2/M transition, prevents activation of cyclin B-associated kinase, and prohibits entry of the surviving cells into mitosis. All of these consequences of the inhibition of PI 3-kinase are relieved by expression of a constitutively active form of PKB (caPKB), indicating that PKB plays a role in regulation of the G2/M phase. Inhibition of PI 3-kinase results in activation of Chk1, whereas caPKB inhibits the ability of Chk1 to become activated in response to treatment with hydroxyurea. Preliminary data show that PKB phosphorylates the Chk1 polypeptide in vitro on serine 280. These results not only implicate PKB activity in transition through the G2/M stage of the cell cycle, but they also suggest the existence of crosstalk between the PI 3-kinase pathway and the key regulators of the DNA damage checkpoint machinery.

AHNAK is a ubiquitously expressed giant phosphoprotein that was initially identified as a gene product subject to transcriptional repression in neuroblastoma. AHNAK is predominantly nuclear in cells of nonepithelial origin, but is cytoplasmic or associated with plasma membrane in epithelial cells. In this study we show that the extranuclear localization of AHNAK in epithelial cells depends on the formation of cell–cell contacts. We show that AHNAK is a phosphorylation substrate of protein kinase B (PKB) in vitro and in vivo. Nuclear exclusion of AHNAK is mediated through a nuclear export signal (NES) in a manner that depends on the phosphorylation of serine 5535 of AHNAK by PKB, a process that also plays a major role in determining extranuclear localization of AHNAK. AHNAK is a new PKB substrate whose function, though unknown, is likely to be regulated by its localization, which is in turn regulated by PKB.

Some prion protein mutations create anchorless molecules that cause Gerstmann–Sträussler–Scheinker (GSS) disease. To model GSS, we generated transgenic mice expressing cellular prion protein (PrP C ) lacking the glycosylphosphatidyl inositol (GPI) anchor, denoted PrP(ΔGPI). Mice overexpressing PrP(ΔGPI) developed a late-onset, spontaneous neurologic dysfunction characterized by widespread amyloid deposition in the brain and the presence of a short protease-resistant PrP fragment similar to those found in GSS patients. In Tg(PrP,ΔGPI) mice, disease onset could be accelerated either by inoculation with brain homogenate prepared from spontaneously ill animals or by coexpression of membrane-anchored, full-length PrP C . In contrast, coexpression of N-terminally truncated PrP(Δ23–88) did not affect disease progression. Remarkably, disease from ill Tg(PrP,ΔGPI) mice transmitted to mice expressing wild-type PrP C , indicating the spontaneous generation of prions.

Doppel (Dpl) protein is a paralog of the prion protein (PrP) that shares 25% sequence similarity with the C-terminus of PrP, a common N-glycosylation site and a C-terminal signal peptide for attachment of a glycosylphophatidyl inositol anchor. Whereas PrPC is highly expressed in the central nervous system (CNS), Dpl is detected mostly in testes and its ectopic expression in the CNS leads to ataxia as well as Purkinje and granule cell degeneration in the cerebellum. The mechanism through which Dpl induces neurotoxicity is still debated. In the present work, primary neuronal cultures derived from postnatal cerebellar granule cells of wild-type and PrP-knockout FVB mice were used in order to investigate the molecular events that occur upon exposure to Dpl. Treatment of cultured cerebellar neurons with recombinant Dpl produced apoptosis that could be prevented by PrP co-incubation. When primary neuronal cultures from Bax-deficient mice were incubated with Dpl, no apoptosis was observed, suggesting an important role of Bax in triggering neurodegeneration. Similarly, cell survival increased when recDpl-treated cells were incubated with an inhibitor of caspase-3, which mediates apoptosis in mammalian cells. Together, our findings raise the possibility that Bax and caspase-3 feature in Dpl-mediated apoptosis.