Abstract
BackgroundUbiquitin E3 ligase CUL4A plays important oncogenic roles in the development of cancers. DTL, one of the CUL4-DDB1 associated factors (DCAFs), may involve in the process of cancer development. Programmed cell death 4 (PDCD4) is a tumor suppressor gene involved in cell apoptosis, transformation, invasion and tumor progression.MethodsAffinity-purification mass spectrometry was used to identify potential DTL interaction proteins. Co-immunoprecipitation (Co-IP) was performed to verify protein interaction between DTL and PDCD4. mRNA levels in cancer cells and tissues were detected by Quantitative real-time PCR. Lentivirus was used to establish stable overexpression and knocking down cell lines for DTL and PDCD4. Transwell and wound healing assays were used to determine migration ability of cancer cells. Matrigel assay was used to determine invasion ability of cancer cells. MTT and colony formation assays were used to evaluate proliferation of cancer cells.ResultsIn this study, programmed cell death 4 (PDCD4) was identified as a potential substrate of DTL. Co-IP and immunofluorescence assays further confirmed the interaction between DTL and PDCD4. Moreover, DTL overexpression decreased the protein level and accelerated the degradation rate of PDCD4. Through in vitro ubiquitination experiment, we proved that PDCD4 was degraded by DTL through ubiquitination. Clinically DTL was significantly up-regulated in cancer tissues than that in normal tissues. The survival curves showed that cancer patients with higher DTL expression owned lower survival rate. Functional experiments showed that DTL not only enhanced the proliferation and migration abilities of cancer cells, but also promoted the tumorigenesis in nude mice. Rescued experiment results demonstrated that silencing PDCD4 simultaneous with DTL recovered the phenotypes defect caused by DTL knocking down.ConclusionsOur results elucidated that DTL enhanced the motility and proliferation of cancer cells through degrading PDCD4 to promote the development of cancers.
Highlights
Ubiquitin E3 ligase CUL4A plays important oncogenic roles in the development of cancers
Denticleless E3 ubiquitin protein ligase homolog (DTL) combines directly with Programmed cell death 4 (PDCD4) To investigate how DTL exerting its functions in cancer cells, proteins interacted with DTL were analyzed using immunoaffinity chromatography
Co-IP demonstrated that CUL4A could interact with PDCD4 (Fig. 1e), and knockdown of DTL weakened the interaction of CUL4A and PDCD4 (Fig. 1f)
Summary
Ubiquitin E3 ligase CUL4A plays important oncogenic roles in the development of cancers. DTL, one of the CUL4-DDB1 associated factors (DCAFs), may involve in the process of cancer development. The ubiquitin-proteasome proteolysis system responsible for degrading proteins is involved in most cellular processes [1]. Cullin protein family containing conserved cullin homology domain is a major type of E3 ligase [4, 5]. DTL, called CDT2, DCAF2 or RAMP, is one of the DCAFs containing seven WD40 domains in its N-terminal. DTL is first identified as retinoic acid-regulated nuclear matrixassociated protein (RAMP) since it is down-regulated during RA-induced differentiation of NT2 cells [7]. The roles of DTL in regulating DNA replication and cell cycle were well-illustrated so far [8]. Studies in yeast revealed that DTL deletion severely slowed down S-phase
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