Cho, Hee Jun
(Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
,
Ryu, Ki-Jun
(Division of Applied Life Science, Research Institute of Life Sciences, Gyeongsang National University)
,
Baek, Kyoung Eun
(Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
,
Lim, Jeewon
(Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
,
Kim, Taeyoung
(Division of Applied Life Science, Research Institute of Life Sciences, Gyeongsang National University)
,
Song, Chae Yeong
(Division of Applied Life Science, Research Institute of Life Sciences, Gyeongsang National University)
,
Yoo, Jiyun
(Division of Applied Life Science, Research Institute of Life Sciences, Gyeongsang National University)
,
Lee, Hee Gu
(Immunotherapy Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays important roles in numerous cellular processes, including cell motility, adhesion, and proliferation, by regulating the activity of Rho GTPases. Its expression is altered in various human cancers and is associated with malignant progres...
Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays important roles in numerous cellular processes, including cell motility, adhesion, and proliferation, by regulating the activity of Rho GTPases. Its expression is altered in various human cancers and is associated with malignant progression. Here, we show that RhoGDI1 interacts with Cullin 3 (CUL3), a scaffold protein for E3 ubiquitin ligase complexes. Ectopic expression of CUL3 increases the ubiquitination of RhoGDI1. Furthermore, potassium channel tetramerization domain containing 5 (KCTD5) also binds to RhoGDI1 and increases its interaction with CUL3. Ectopic expression of KCTD5 increases the ubiquitination of RhoGDI1, whereas its knockdown by RNA interference has the opposite effect. Depletion of KCTD5 or expression of dominant-negative CUL3 (DN-CUL3) enhances the stability of RhoGDI1. Our findings reveal a previously unknown mechanism for controlling RhoGDI1 degradation that involves a CUL3/KCTD5 ubiquitin ligase complex.
Rho guanine nucleotide dissociation inhibitor 1 (RhoGDI1) plays important roles in numerous cellular processes, including cell motility, adhesion, and proliferation, by regulating the activity of Rho GTPases. Its expression is altered in various human cancers and is associated with malignant progression. Here, we show that RhoGDI1 interacts with Cullin 3 (CUL3), a scaffold protein for E3 ubiquitin ligase complexes. Ectopic expression of CUL3 increases the ubiquitination of RhoGDI1. Furthermore, potassium channel tetramerization domain containing 5 (KCTD5) also binds to RhoGDI1 and increases its interaction with CUL3. Ectopic expression of KCTD5 increases the ubiquitination of RhoGDI1, whereas its knockdown by RNA interference has the opposite effect. Depletion of KCTD5 or expression of dominant-negative CUL3 (DN-CUL3) enhances the stability of RhoGDI1. Our findings reveal a previously unknown mechanism for controlling RhoGDI1 degradation that involves a CUL3/KCTD5 ubiquitin ligase complex.
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문제 정의
In this study, we report a novel molecular mechanism for the post-translational regulation of RhoGDI1 expression that is dependent on CUL3-mediated ubiquitination. KCTD5 seems to also be an important part of this mechanism.
RhoGDI1 is also associated with cancer cell migration, invasion, metastasis, and chemoresistance via regulation of Rho GTPase activity [22]. Therefore, our future efforts will focus on verifying the effect of the CUL3/KCTD5-mediated regulation of RhoGDI1 stability on cancer progression.
가설 설정
HEK293T cells were co-transfected Myc-CUL3 with Flag-RhoGDI1 or Flag-RhoGDI2. (A) Cell lysates were immunoprecipitated with anti-Myc antibody. Immunoprecipitates and total lysates were immunoblotted with anti-Myc and anti-Flag antibodies.
Immunoprecipitates and total lysates were immunoblotted with anti-Myc and anti-Flag antibodies. (B) Cell lysates were immunoprecipitated with anti-Flag antibody. Immunoprecipitates and total lysates were immunoblotted with anti-Myc and anti-Flag antibodies.
Based on the collaboration of CUL3 and KCTD5 in RhoGDI1 ubiquitination, we hypothesized that these proteins might interact within a ubiquitin ligase complex that marks RhoGDI1 for degradation. To determine the effect of CUL3 and KCTD5 on RhoGDI1 stability, we monitored the levels of the latter in CHX-treated HEK293T cells overexpressing HA-ubiquitin and Flag-RhoGDI1.
제안 방법
Based on the collaboration of CUL3 and KCTD5 in RhoGDI1 ubiquitination, we hypothesized that these proteins might interact within a ubiquitin ligase complex that marks RhoGDI1 for degradation. To determine the effect of CUL3 and KCTD5 on RhoGDI1 stability, we monitored the levels of the latter in CHX-treated HEK293T cells overexpressing HA-ubiquitin and Flag-RhoGDI1. As seen in Fig.
대상 데이터
HEK293T cells were obtained from the American Type Culture Collection (USA). Cells were cultured at 37℃ in a humidified 5% CO2 incubator in DMEM (Invitrogen, USA) supplemented with 10% FBS and antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin).
The KCTD5 siRNA (54442-1) and the AccuTarget Negative Control siRNA (siCon) were obtained from Bioneer (Korea). Transient transfection of each siRNA was accomplished using Lipofectamine RNAiMAX (Invitrogen) according to the instructions of the manufacturer.
데이터처리
Differences were assessed using Student’s t-tests, with p < 0.05 considered statistically significant.
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