Figure 2.
Figure 2. Mapping of the ubiquitination and degradation pattern of CDK2. (A) The stability of CDK2 protein was measured by western blotting. NB4 and COS-7 cells were cultured with 10 µg/mL CHX for indicated times in the presence of vehicle or MG132 (1 or 10 µM). (B) Ubiquitination of CDK2 in COS-7 cells. COS-7 cells were cotransfected with pCMV-CDK2-HA and pEBB-His-C1-Ub for 48 hours, and 10 μM MG132 was added for the final 8 hours of culture. Ubiquitination was determined by immunoprecipitation with HA followed by western blotting with His antibody. (C) The ubiquitination of CDK2 in the RRL cell-free system. Cell extracts from COS-7 cells transfected with the pCMV or pCMV-CDK2-HA plasmid were subjected to anti-HA immunoprecipitation, followed by incubation with or without RRL at 30°C for 2 hours. (D) The effect of k48r and k63r ubiquitin on the ubiquitination of CDK2 in COS-7 cells. Ubiquitination was determined by immunoprecipitation with HA followed by western blotting with His antibody. PI: preimmune IgG; Δ, IgG heavy chain. (E) The turnover rates of various CDK2 mutants whose single lysine (K) was replaced with arginine (R) were measured by western blotting. COS-7 cells transfected with different CDK2 mutants were cultured with 10 µg/mL CHX for 6 hours. (F) The turnover rates of various CDK2 mutants carrying only 1 of the lysine as indicated. CDK2-9R is a mutant whose 9 lysine (Lys-20, Lys-24, Lys-33, Lys-129, Lys-142, Lys-237, Lys-250, Lys-273, and Lys-287) were replaced with arginine. These 9 mutants were derived from CDK2-9R by adding back individual lysine as indicated. (G) The decreased rates of various CDK2 mutants carrying 2 to 3 KR replacements. Diagrams of WT CDK2 with predicted ubiquitination sites and various CDK2 mutant constructs are shown. (H) The effect of KLHL6 on the ubiquitination of CDK2. HeLa cells were overexpressed with His-Ub, CDK2-HA, and Flag-KLHL6 for 48 hours, and then ubiquitination was determined by immunoprecipitation with HA followed by western blotting with His antibody. (I) The interaction between CDK2 and KLHL6 was detected by immunoprecipitation. HeLa cells were overexpressed with CDK2-HA and Flag-KLHL6 for 48 hours. After immunoprecipitation with HA their interaction were detected by western blotting with Flag antibody. (J) The effect of KLHL6 on the degradation of endogenous CDK2 protein as determined by western blotting. HeLa cells were overexpressed with Flag-KLHL6 followed with treatment with 10 μg/mL CHX for the indicated times. (K) The effect of KLHL6 on the degradation of WT CDK2 and CDK2 (M-2R) mutant. HeLa cells cotransfected with CDK2-HA (WT or M-2R) and Flag-KLHL6 plasmids were treated with 10 μg/mL CHX for the indicated times, and the exogenous CDK2 levels were determined by western blotting with HA antibody. (L) The effect of shKLHL6 on the degradation of endogenous CDK2 protein. HeLa cells were transfected with shKLHL6 #1 plasmids followed by CHX treatment of the indicated times, and the protein levels of CDK2 were determined by western blotting. HA, hemagglutinin; IP, immunoprecipitation; l.e., long exposure; s.e., short exposure. Data are representative of at least 3 individual experiments; 1 representative image is shown.

Mapping of the ubiquitination and degradation pattern of CDK2. (A) The stability of CDK2 protein was measured by western blotting. NB4 and COS-7 cells were cultured with 10 µg/mL CHX for indicated times in the presence of vehicle or MG132 (1 or 10 µM). (B) Ubiquitination of CDK2 in COS-7 cells. COS-7 cells were cotransfected with pCMV-CDK2-HA and pEBB-His-C1-Ub for 48 hours, and 10 μM MG132 was added for the final 8 hours of culture. Ubiquitination was determined by immunoprecipitation with HA followed by western blotting with His antibody. (C) The ubiquitination of CDK2 in the RRL cell-free system. Cell extracts from COS-7 cells transfected with the pCMV or pCMV-CDK2-HA plasmid were subjected to anti-HA immunoprecipitation, followed by incubation with or without RRL at 30°C for 2 hours. (D) The effect of k48r and k63r ubiquitin on the ubiquitination of CDK2 in COS-7 cells. Ubiquitination was determined by immunoprecipitation with HA followed by western blotting with His antibody. PI: preimmune IgG; Δ, IgG heavy chain. (E) The turnover rates of various CDK2 mutants whose single lysine (K) was replaced with arginine (R) were measured by western blotting. COS-7 cells transfected with different CDK2 mutants were cultured with 10 µg/mL CHX for 6 hours. (F) The turnover rates of various CDK2 mutants carrying only 1 of the lysine as indicated. CDK2-9R is a mutant whose 9 lysine (Lys-20, Lys-24, Lys-33, Lys-129, Lys-142, Lys-237, Lys-250, Lys-273, and Lys-287) were replaced with arginine. These 9 mutants were derived from CDK2-9R by adding back individual lysine as indicated. (G) The decreased rates of various CDK2 mutants carrying 2 to 3 KR replacements. Diagrams of WT CDK2 with predicted ubiquitination sites and various CDK2 mutant constructs are shown. (H) The effect of KLHL6 on the ubiquitination of CDK2. HeLa cells were overexpressed with His-Ub, CDK2-HA, and Flag-KLHL6 for 48 hours, and then ubiquitination was determined by immunoprecipitation with HA followed by western blotting with His antibody. (I) The interaction between CDK2 and KLHL6 was detected by immunoprecipitation. HeLa cells were overexpressed with CDK2-HA and Flag-KLHL6 for 48 hours. After immunoprecipitation with HA their interaction were detected by western blotting with Flag antibody. (J) The effect of KLHL6 on the degradation of endogenous CDK2 protein as determined by western blotting. HeLa cells were overexpressed with Flag-KLHL6 followed with treatment with 10 μg/mL CHX for the indicated times. (K) The effect of KLHL6 on the degradation of WT CDK2 and CDK2 (M-2R) mutant. HeLa cells cotransfected with CDK2-HA (WT or M-2R) and Flag-KLHL6 plasmids were treated with 10 μg/mL CHX for the indicated times, and the exogenous CDK2 levels were determined by western blotting with HA antibody. (L) The effect of shKLHL6 on the degradation of endogenous CDK2 protein. HeLa cells were transfected with shKLHL6 #1 plasmids followed by CHX treatment of the indicated times, and the protein levels of CDK2 were determined by western blotting. HA, hemagglutinin; IP, immunoprecipitation; l.e., long exposure; s.e., short exposure. Data are representative of at least 3 individual experiments; 1 representative image is shown.

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