Normal p53 Function in Primary Cells Deficient for Siah Genes

Abstract

Overexpression studies have suggested that Siah1 proteins may act as effectors of p53-mediated cellular responses and as regulators of mitotic progression. We have tested these hypotheses using Siah gene knockout mice. Siah1a and Siah1b were not induced by activation of endogenous p53 in tissues, primary murine embryonic fibroblasts (MEFs) or thymocytes. Furthermore, primary MEFs lacking Siah1a, Siah1b, Siah2, or both Siah2 and Siah1a displayed normal cell cycle progression, proliferation, p53-mediated senescence, and G 1 phase cell cycle arrest. Primary thymocytes deficient for Siah1a, Siah2, or both Siah2 and Siah1a, E1A-transformed MEFs lacking Siah1a, Siah1b, or Siah2, and Siah1b-null ES cells all underwent normal p53-mediated apoptosis. Finally, inhibition of Siah1b expression in Siah2 Siah1a double-mutant cells failed to inhibit cell division, p53-mediated induction of p21 expression, or cell cycle arrest. Our loss-of-function experiments do not support a general role for Siah genes in p53-mediated responses or mitosis.p53 acts as a tumor suppressor by inducing cell cycle arrest, apoptosis, and senescence in response to cellular stresses such as DNA damage or oncogene activation. These effects are mediated largely through the function of p53 as a transcriptional activator or repressor (5, 25). A diverse range of p53 target genes have been identified, for example, p21, an important mediator of G 1 phase cell cycle arrest and cellular senescence (10), and several proapoptotic genes including Noxa and Bax (53). However, understanding of the full range of p53 effectors is incomplete and it is likely that multiple signaling pathways are involved in determining cellular responses to p53 activation.Recent studies have proposed that Siah1, a member of the Siah family of E3 ubiquitin ligases, may act as a downstream effector of p53. Overexpression of p53 induces the transcriptional activation of Siah1 family genes in a variety of mammalian cell lines (1,20,32,35,43,44,47), and overexpression of Siah1 can mimic the effects of p53 activation and induce cell cycle arrest or apoptosis (37,43,44).A molecular mechanism by which Siah1 proteins may mediate p53 function has been proposed. Human SIAH1 functions as a component of an E3 ubiquitin ligase complex including Siah-interacting protein, Skp1, and the F-box protein Ebi, which is proposed to target ␤-catenin for ubiquitin-mediated degradation in response to activation of p53 (32,36,42). Importantly, overexpression of either p53 or SIAH1 can induce ␤-catenin degradation, and this is blocked by coexpression of an N-terminally truncated SIAH1 protein lacking the RING domain. By this model, p53-mediated induction of SIAH1 following DNA damage induces degradation of ␤-catenin independently of the GSK3␤-mediated degradation pathway. Since overexpression of ␤-catenin promotes cell cycle progression and inhibits cell cycle arrest induced by gamma irradiation (41), SIAH1-mediated ␤-catenin degradation may contribute to p53-dependent cell cycle arrest. Interestingly, accumulatio...

Siah Proteins: Novel Drug Targets in the Ras and Hypoxia Pathways

“…Interestingly, contrary to previous studies linking Siah proteins with p53 signaling, Siah expression has been shown to be stable in mouse tissues and primary cells following activation of endogenous p53 by genotoxic stress (24). Importantly, p53 signaling, including up-regulation of p21, seemed normal in primary fibroblasts that lacked Siah genes (24). Recently, Siah1 has been reported as central to a conserved module involved in the innate immune response in Caenorhabditis elegans (25) and has been previously associated with immune signaling involving TRAF2 (6) and TNF-α (5).…”

“…Although Siah2 null mice are outwardly normal in appearance (23), compound Siah1a/Siah2 mutation is lethal, implying a level of functional redundancy (23 is no apparent increase in tumor incidence in Siah1a or Siah2 knockout mice, as might be expected with deletion of a tumor suppressor protein. Interestingly, contrary to previous studies linking Siah proteins with p53 signaling, Siah expression has been shown to be stable in mouse tissues and primary cells following activation of endogenous p53 by genotoxic stress (24). Importantly, p53 signaling, including up-regulation of p21, seemed normal in primary fibroblasts that lacked Siah genes (24).…”

The Coiled-coil Domain Is the Structural Determinant for Mammalian Homologues of Drosophila Sina-mediated Degradation of Promyelocytic Leukemia Protein and Other Tripartite Motif Proteins by the Proteasome

“…SIAH-1 is transcriptionally induced by p53 in human cell lines and apparently plays a positive role in the p53 stress response (8). Recent results from Siah-1/2 knock out mice suggest that this regulation might differ in murine cells or does not play a major role in vivo, leaving the discussion open on the significance of the Siah/p53 network (9). Nevertheless, the fact that SIAH may then contribute to the down-regulation of PML (which is itself a p53 regulator) (14,47,49) suggests a feedback loop to control p53 activity.…”

Siah Proteins Induce the Epidermal Growth Factor-dependent Degradation of Phospholipase Cϵ

“…Mouse monoclonal antibody of Siah1 was described previously (30). Other antibodies used were: goat polyclonal anti-Siah2 antibody and goat polyclonal anti-AH FIGURE 1.…”

“…Cell Culture-COS-7 cells were grown in DMEM containing 10% bovine calf serum, antibiotics, and glutamine. Wild-type and Siah-deficient MEF cells were grown in DMEM containing 10% fetal bovine serum, 0.02 mM ␤-mercaptoethanol, antibiotics, and glutamine as previously described (30). Cells were grown to ϳ90% confluence for immunoprecipitation and Western blot experiments.…”