PIAS-1 Is a Checkpoint Regulator Which Affects Exit from G₁ and G₂ by Sumoylation of p73

Abstract

p73 is a recently described member of the p53 family, and, like p53, it undergoes a number of posttranslational modifications. Here we show, by yeast two-hybrid screening, pull-down assays, and coimmunoprecipitation, that p73␣, -␤, and -␥ bind to the protein inhibitor of activated STAT-1 (PIAS-1) and that this binding stabilizes p73. PIAS-1 also sumoylates p73␣, although not the C-terminally truncated isoforms p73␤ and -␥, and this requires the RING finger domain of PIAS-1. The ⌬Np73␣ isoform can also bind, and be sumoylated by, PIAS-1. PIAS-1-mediated sumoylation decreases p73 transcriptional activity on several target promoters, such as Bax. p73 is colocalized in the nucleus with PIAS-1, and sumoylated p73 is located exclusively in the nuclear matrix. PIAS-1 is expressed predominantly during S phase, and PIAS-1 overexpression reduces p73-mediated transcription of p21, with a reduction of cells in G 1 and cell cycle reentry. Inhibition of endogenous PIAS-1 by RNA interference reduces the proportion of cells in S phase and induces G 2 arrest. These data suggest that PIAS-1, acting partly through binding and sumoylation of p73, is an important component of the cell cycle machinery.Signal transducer and activator of transcription (STAT) proteins are cytoplasmic transcription factors which, after phosphorylation, translocate to the nucleus, where they regulate expression of STAT-responsive genes (reviewed in references 4 and 14). STAT activity is partly regulated by a family of at least six related proteins, the protein inhibitors of activated STATs (PIAS). Thus, PIAS-1 inhibits DNA binding and gene activation by activated, tyrosine-phosphorylated STAT-1 (24). Another protein, PIASy, also inhibits the transcriptional activity of activated STAT-1, without, however, affecting STAT-1 DNA binding (25). A region located at the carboxy terminus of PIAS-1 directly interacts with STAT-1 dimers, though its interaction with phosphorylated and nonphosphorylated monomers is inhibited by the N-terminal domain (23). Recently, it has become clear that PIAS proteins can also regulate the activity of other transcription factors such as Smads (25), the mineralocorticoid and glucocorticoid receptors (40), and p53 (38).At least part of the mechanism of transcriptional regulation by PIAS proteins depends on their E3 SUMO ligase activity, and PIAS-1 and SUMO-1 colocalize in nuclear granules (19). Thus, PIAS proteins sumoylate activated STAT-1, resulting in reduced STAT-1-mediated transcription (42). Moreover, the RING finger domain of PIAS-1 binds to the C terminus of the tumor suppressor p53 (33) and catalyzes its sumoylation (17), a modification which represses p53 activity on a reporter plasmid containing consensus p53 DNA binding sites (38). However, other studies using the p21 promoter have shown enhanced p53 transcriptional activity after interaction with PIAS-1, although this is independent of the RING finger domain and therefore of p53 sumoylation (26). Two homologues of p53, p63 and p73, have been described. The three proteins ...

Altered expression of prolactin receptor-associated signaling proteins in human breast carcinoma

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p73 regulates autophagy and hepatocellular lipid metabolism through a transcriptional activation of the ATG5 gene

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FLASH is required for histone transcription and S-phase progression

“…Nuclear extracts were prepared by using Cell Lytic Nuclear Extraction kit (Sigma). Immunoprecipitation was performed as described, incubating 3 mg of nuclear extracts with anti-GFP polyclonal antibody, 632460 (BD Clontech) (17). Membranes were probed with the anti-NPAT antibody, 611344 (BD Transduction Laboratories).…”

“…Colony-forming assay was performed on SAOS2 cells transfected with pSUPER-FLASH-1 or pSUPERscrambled together with a pBabe-puro vector for selection as described (17).…”

“…Western blots were performed as described (17) by using the following antibodies: anti-FLASH, M300, sc-9088, lot no. B040 (Santa Cruz Biotechnology), anti-FLASH antibody rabbit SL1133 and SL1134 generated in our laboratory; antiHistone H4, #2592 (Cell Signaling, Beverly, MA); anti-Histone H3 no.…”