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A RIP-like protein, RIP3, has recently been reported that contains an N-terminal kinase domain and a novel Cterminal domain that promotes apoptosis. These experiments further characterize RIP3-mediated apoptosis and NF-U UB activation. Northern blots indicate that rip3 mRNA displays a restricted pattern of expression including regions of the adult central nervous system. The rip3 gene was localized by fluorescent in situ hybridization to human chromosome 14q11.2, a region frequently altered in several types of neoplasia. RIP3-mediated apoptosis was inhibited by Bcl-2, Bcl-x L , dominant-negative FADD, as well as the general caspase inhibitor Z-VAD. Further dissection of caspase involvement in RIP3-induced apoptosis indicated inhibition by the more specific inhibitors Z-DEVD (caspase-3, -6, -7, -8, and -10) and Z-VDVAD (caspase-2). However, caspase-1, -6, -8 and -9 inhibitors had little or no effect on RIP3-mediated apoptosis. Mutational analysis of RIP3 revealed that the C-terminus of RIP3 contributed to its apoptotic activity. This region is similar, but distinct, to the death domain found in many pro-apoptotic receptors and adapter proteins, including FAS, FADD, TNFR1, and RIP. Furthermore, point mutations of RIP3 at amino acids conserved among death domains, abrogated its apoptotic activity. RIP3 was localized by immunofluorescence to the mitochondrion and may play a key role in the mitochondrial disruptions often associated with apoptosis.z 2000 Federation of European Biochemical Societies.

Previously, we established that persistent upregulation of c-fos expression preceded kainic acid (KA)-induced neuronal death in mice. To discriminate between events that are products of the seizures elicited by KA and those that are specifically associated with its neurotoxic actions, we have examined the expression of cellular immediate-early genes (cIEGs) following KA or pentylenetetrazol (PTZ) treatment in c-fos-lacZ transgenic rats. While both chemoconvulsants elicit seizures, only KA causes selective neuronal death. Following treatment of transgenic rats with KA there was a protracted expression of Fos-lacZ that lasted for 2-3 d. In contrast, PTZ elicited a transient increase in the transgene product that lasted about 6 hr. Normally, Fos and Fos-lacZ were detected only in neuronal nuclei. However, 6 hr following kainic acid (but not PTZ) administration, beta-galactosidase activity appeared in the cytoplasm of neurons within vulnerable regions (as determined by the terminal transferase biotinylated-UTP nick end labeling (TUNEL) procedure). Like c-fos, transcripts for other cIEGs were elevated for longer periods in the KA-treated rat hippocampus. In addition, fra-1 and fra-2 were only induced in the KA-treated rat. These changes in mRNA levels were paralleled by a sustained increase in AP-1 DNA binding activity. Thus, quantitative and qualitative changes in AP-1 DNA binding complexes accompany neurotoxic cell death that are not observed following seizures.

Highlights d Syntaxin 17 functions during autophagy initiation and bulk cargo sequestration d TBK1 phosphorylates syntaxin 17 at Ser202 (Stx17 pS202) d Stx17 pS202 translocates from Golgi to pre-autophagosomal structure upon starvation d Stx17 pS202 controls formation of FIP200-ATG13 preautophagosomal structures

The tumor necrosis factor (TNF) receptor family are ligand-regulated transmembrane proteins that mediate apoptosis as well as activation of the transcription factor NF-kB. Exogenous expression of DR6, a recently identi®ed member of the TNF receptor family, induced apoptosis in untransformed or tumor-derived cells and the apoptotic function of DR6 was inhibited by coexpression of Bcl-2, Bcl-x L or the inhibitor-of-apoptosis (IAP) family member, survivin. Expression of a dominant negative mutant of FADD failed to protect from DR6-mediated apoptosis indicating that unlike TNFR1 and Fas, DR6 induced apoptosis via a FADDindependent mechanism. Despite the ability of exogenous DR6 expression to induce apoptosis, DR6 mRNA and protein were found to be elevated in prostate tumor cell lines and in advanced stages of prostate cancer. Analysis of several anti-apoptotic proteins revealed that Bcl-x L levels and serine 32 phosphorylation of IkB, the natural inhibitor of NF-kB, were similarly elevated in cells expressing high levels of DR6, suggesting that NF-kBregulated survival proteins may protect from DR6-induced apoptosis and that DR6 is a target of NF-kB regulation. Treatment of LnCAP cells with TNF-a resulted in increases in both DR6 mRNA and protein levels, and this induction was suppressed by inhibitors of NF-kB. Similarly, treatment of cells expressing high levels of DR6 with indomethacin and ibuprofen, compounds also known to perturb NF-kB function, resulted in a dose-dependent decrease in DR6 protein and mRNA levels. These results demonstrate that TNF-a signaling induces the expression of a member of its own receptor family through activation of NF-kB. Oncogene (2001) 20, 7965 ± 7975.

Highlights d Autophagy and the autophagy-inducing kinase ULK1 affect TNF-induced cell death d Ablation of ULK1 expression or activity enhances complex IIb/necrosome formation d The necroptosis-regulating kinase RIPK1 is a substrate of ULK1 d ULK1-dependent phosphorylation of RIPK1 at Ser357 reduces TNF-induced cell death

Background: Although Mcl-1 is normally subject to rapid turnover via a phosphodegron at Thr-163/Ser-159 and other pathways, sustained expression promotes viability/chemoresistance in cancer cells.