The evolution of mitogenic pathways has led to the parallel requirement for negative control mechanisms, which prevent aberrant growth and the development of cancer. Principally, such negative control mechanisms are represented by tumor suppressor genes, which normally act to constrain cell proliferation (Macleod, K. 2000. Curr. Opin. Genet. Dev. 10:81–93). Tuberous sclerosis complex (TSC) is an autosomal-dominant genetic disorder, characterized by mutations in either TSC1 or TSC2, whose gene products hamartin (TSC1) and tuberin (TSC2) constitute a putative tumor suppressor complex (TSC1-2; van Slegtenhorst, M., M. Nellist, B. Nagelkerken, J. Cheadle, R. Snell, A. van den Ouweland, A. Reuser, J. Sampson, D. Halley, and P. van der Sluijs. 1998. Hum. Mol. Genet. 7:1053–1057). Little is known with regard to the oncogenic target of TSC1-2, however recent genetic studies in Drosophila have shown that S6 kinase (S6K) is epistatically dominant to TSC1-2 (Tapon, N., N. Ito, B.J. Dickson, J.E. Treisman, and I.K. Hariharan. 2001. Cell. 105:345–355; Potter, C.J., H. Huang, and T. Xu. 2001. Cell. 105:357–368). Here we show that loss of TSC2 function in mammalian cells leads to constitutive S6K1 activation, whereas ectopic expression of TSC1-2 blocks this response. Although activation of wild-type S6K1 and cell proliferation in TSC2-deficient cells is dependent on the mammalian target of rapamycin (mTOR), by using an S6K1 variant (GST-ΔC-S6K1), which is uncoupled from mTOR signaling, we demonstrate that TSC1-2 does not inhibit S6K1 via mTOR. Instead, we show by using wortmannin and dominant interfering alleles of phosphatidylinositide-3-OH kinase (PI3K) that increased S6K1 activation is contingent upon the suppression of TSC2 function by PI3K in normal cells and is PI3K independent in TSC2-deficient cells.

The delicate balance between the synthesis and the degradation of proteins ensures cellular homeostasis. Proteases act in an irreversible manner and therefore have to be strictly regulated. The ubiquitin-proteasome system (UPS) is a major pathway for the proteolytic degradation of cellular proteins. As dysregulation of the UPS is observed in most cancers including leukemia, the UPS is a valid target for therapeutic intervention strategies. Ubiquitin-ligases selectively bind substrates to target them for poly-ubiquitinylation and proteasomal degradation. Therefore, pharmacological modulation of these proteins could allow a specific level of control. Increasing evidence accumulates that ubiquitin-ligases termed mammalian seven in absentia homologs (SIAHs) are not only critical for the pathogenesis of solid tumors but also for leukemogenesis. However, the relevance and therapeutic potential of SIAH-dependent processes has not been fully elucidated. Here, we summarize functions of SIAH ubiquitin-ligases in leukemias, how they select leukemia-relevant substrates for proteasomal degradation, and how the expression and activity of SIAH1 and SIAH2 can be modulated in vivo. We also discuss that epigenetic drugs belonging to the group of histone deacetylase inhibitors induce SIAH-dependent proteasomal degradation to accelerate the turnover of leukemogenic proteins. In addition, our review highlights potential areas for future research on SIAH proteins.

SummaryThe Wilms' tumor suppressor protein WT1 functions as a transcriptional regulator of genes controlling growth, apoptosis, and differentiation. It has become clear that WT1 can act as an oncogene in many tumors, primarily through the inhibition of apoptosis. Here, we identify the serine protease HtrA2 as a WT1 binding partner and find that it cleaves WT1 at multiple sites following the treatment of cells with cytotoxic drugs. Ablation of HtrA2 activity either by chemical inhibitor or by siRNA prevents the proteolysis of WT1 under apoptotic conditions. Moreover, the apoptosis-dependent cleavage of WT1 is defective in HtrA2 knockout cells. Proteolysis of WT1 by HtrA2 causes the removal of WT1 from its binding sites at gene promoters, leading to alterations in gene regulation that enhance apoptosis. Our findings provide insights into the function of HtrA2 in the regulation of apoptosis and the oncogenic activities of WT1.

is a post-translational modification that regulates various physiological processes, including DNA damage repair, gene transcription and signal transduction. Intracellular ADP-ribosyltransferases (ARTDs or PARPs) modify their substrates either by poly-or mono-ADP-ribosylation. Previously we identified ARTD10 (formerly PARP10) as a mono-ADP-ribosyltransferase, and observed that exogenous ARTD10 but not ARTD10-G888W, a catalytically inactive mutant, interferes with cell proliferation. To expand on this observation, we established cell lines with inducible ARTD10 or ARTD10-G888W. Consistent with our previous findings, induction of the wild-type protein but not the mutant inhibited cell proliferation, primarily by inducing apoptosis. During apoptosis, ARTD10 itself was targeted by caspases. We mapped the major cleavage site at EIAMD406↓S, a sequence that was preferentially recognized by caspase-6. Caspase-dependent cleavage inhibited the pro-apoptotic activity of ARTD10, as ARTD10(1-406) and ARTD10(407-1025), either alone or together, were unable to induce apoptosis, despite catalytic activity of the latter. Deletion of the N-terminal RNA recognition motif in ARTD10(257-1025) also resulted in loss of pro-apoptotic activity. Thus our findings indicate that the RNA recognition motif contributes to the pro-apoptotic effect, together with the catalytic domain. We suggest that these two domains must be physically linked to stimulate apoptosis, possibly targeting ARTD10 through the RNA recognition motif to specific substrates that control cell death. Moreover, we established that knockdown of ARTD10 reduced apoptosis in response to DNA-damaging agents. Together, these findings indicate that ARTD10 is involved in the regulation of apoptosis, and that, once apoptosis is activated, ARTD10 is cleaved as part of negative feedback regulation. Structured digital abstractCaspase-8 cleaves ARTD10 by protease assay (View interaction) Caspase-6 cleaves ARTD10 by protease assay (View interaction) Abbreviations ART, ADP-ribosyltransferase; ARTD, ADP-ribosyltransferase diphtheria toxin-like; FACS, fluorescence-activated cell sorting; HA, hemagglutinin; PI, propidium iodide; RRM, RNA recognition motif.