p14ARF Interacts with the SUMO-Conjugating Enzyme Ubc9 and Promotes the Sumoylation of Its Binding Partners

Abstract

Bcl-2 phosphorylation is a normal physiological process occurring at mitosis or during mitotic arrest induced by microtubule damaging agents. The consequences of Bcl-2 phosphorylation on its function are still controversial. To better understand the role of Bcl-2 phosphorylation in mitosis, we studied the subcellular localization of phosphorylated forms of Bcl-2. Immunofluorescence experiments performed in synchronized HeLa cells indicate for the first time that mitotic phosphorylated forms of Bcl-2 can be detected in nuclear structures in prophase cells together with nucleolin and Ki-67. In later mitotic stages, as previously described, phosphorylated forms of Bcl-2 are localized on mitotic chromosomes. In addition, we demonstrate that Bcl-2 in these structures is at least in part phosphorylated on the T56 residue. Then, coimmunoprecipitation experiments reveal that, in cells synchronized at the onset of mitosis, Bcl-2 is present in a complex with nucleolin, cdc2 kinase and PP1 phosphatase. Taken together, these data further support the idea that Bcl-2 could have a new function at mitosis.

Sumoylation induced by the Arf tumor suppressor: A p53-independent function

“…Human p14 ARF has been reported to bind to several other proteins, including the WRN helicase, E2F1, c-Myc, topoisomerase I, HIF-1␣, the adenovirus E1A-regulated transcriptional repressor p120 E4F , and the HIV Tat-binding protein 1 (12,13,16,17,21,22,41). With the exception of c-Myc, which has not been tested, these proteins can undergo sumoylation when cotransfected with p14 ARF and His 6 -SUMO-1 into human tumor cell lines.…”

“…For example, might p19 Arf serve as a specificity factor that bridges Ubc9-containing complexes to Arf-binding proteins? Woods et al (21) stated that robust SUMO-1 conjugation of WRN could be observed in an in vitro reconstitution reaction in the presence of the SUMO E1͞E2 enzymes but indicated that addition of recombinant p14 ARF had no effect, even at limiting concentrations of Ubc9 with which tagged p14 ARF can coprecipitate when overexpressed (22). An inability to stimulate sumoylation in vitro with recombinant p14 ARF may not be surprising given the fact that Arf proteins are highly basic, bind nonspecifically to many acidic proteins and to nucleic acids, and normally reside in ''buffered'' higher-molecular-mass complexes in living cells (40).…”

“…Enforced expression of p14 ARF in human cells promotes sumoylation of several cotransfected cellular proteins, including Hdm2 (19,20), the Werner helicase (WRN) (21), and the transcription factors E2F-1 and HIF-1␣ (22). Covalent addition of the small ubiquitin-like modifiers (SUMO-1, SUMO-2, and SUMO-3) to the -amino groups of certain lysine residues in protein substrates is carried out by a single E1 enzyme (the SUMO-1-activating enzyme 1͞2 heterodimer also known as Aos1͞Uba2) and a single E2 enzyme (Ubc9) in a process analogous to ubiquitination (23)(24)(25).…”

A Novel Link Between SUMO Modification and Cancer Metastasis

“…[24][25][26][27][28][29][30] Although ubiquitin was discovered more than 25 years ago, SUMO was discovered less than 10 years ago. However, this late entrant is becoming an important molecule in cell biology and cancer biology.…”

“…[24][25][26][27][28][29][30][31][32] The initial maturation step of SUMO requires C-terminal processing, leading to the exposure of a GG motif at its C-terminal end, allowing the conjugation process to occur. Following this step, heterodimeric E1 enzymes, SAE1/ SAE2, catalyze the ATP-dependent activation step.…”