Abstract
Tumor suppressor RASSF1A (RAS association domain family 1, isoform A) is known to play an important role in regulation of mitosis; however, little is known about how RASSF1A is regulated during the mitotic phase of the cell cycle. In the present study, we have identified Cullin-4A (CUL4A) as a novel E3 ligase for RASSF1A. Our results demonstrate that DNA damage-binding protein 1 (DDB1) functions as a substrate adaptor that directly interacts with RASSF1A and bridges RASSF1A to the CUL4A E3 ligase complex. Depletion of DDB1 also diminishes intracellular interactions between RASSF1A and CUL4A. Our results also show that RASSF1A interacts with DDB1 via a region containing amino acids 165-200, and deletion of this region abolishes RASSF1A and DDB1 interactions. We have found that CUL4A depletion results in increased levels of RASSF1A protein due to increased half-life; whereas overexpression of CUL4A and DDB1 markedly enhances RASSF1A protein ubiquitination resulting in reduced RASSF1A levels. We further show that CUL4A-mediated RASSF1A degradation occurs during mitosis, and depletion of CUL4A markedly reverses mitotic-phase-stimulated RASSF1A degradation. We also note that overexpression of CUL4A antagonizes the ability of RASSF1A to induce M-phase cell cycle arrest. Thus, our present study demonstrates that the CUL4A·DDB1 E3 complex is important for regulation of RASSF1A during mitosis, and it may contribute to inactivation of RASSF1A and promoting cell cycle progression.
Highlights
RASSF1A Interacts with DNA damage-binding protein 1 (DDB1)—We have previously shown that RASSF1A affects M-phase of cell cycle progression by modulating the microtubule dynamic and Aurora-A phosphorylates RASSF1A to influence its ability to associate with microtubules and regulate cell cycle [8, 17]
We have identified the CUL4A1⁄7DDB1 complex as a novel E3 ligase for tumor suppressor RASSF1A
Our studies demonstrate that DDB1 functions as a substrate adapter; it directly interacts with RASSF1A and bridges RASSF1A to the CUL4A E3 ligase complex
Summary
A number of recent studies including ours have shown that RASSF1A induces cell cycle arrest in both G1/S- and mitotic (M)-phases [8, 11,12,13,14,15,16]. HEK293T cells were independently transfected with GFP-tagged full-length RASSF1A or ⌬165– 200 deletion variant RASSF1A or GFP alone expression vectors, and co-immunoprecipitation assays were performed. HEK293T cells were transfected with HAS-tag vector alone or HA-S-tag RASSF1A, and cell lysates from the transfectants were used for S-protein pulldown assay to investigate RASSF1A-CUL4A interactions.
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