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First published online 1 June 2004
doi: 10.1242/jcs.01086

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Binding of 14-3-3ß but not 14-3-3 controls the cytoplasmic localization of CDC25B: binding site preferences of 14-3-3 subtypes and the subcellular localization of CDC25B

Sanae Uchida¹, Akiko Kuma^1,*, Motoaki Ohtsubo², Mari Shimura³, Masato Hirata⁴, Hitoshi Nakagama⁵, Tsukasa Matsunaga⁶, Yukihito Ishizaka³ and Katsumi Yamashita^1,

¹ Division of Life Science, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma-machi, Kanazawa, 920-1192, Japan
² Institute of Life Science, Kurume University, Aikawa 2432-3, Kurume, 839-0861, Japan
³ Division of Intractable Disease, International Medical Center of Japan, 21-1 Toyama 1-chome, Shinjuku-ku, Tokyo, 162-8655, Japan
⁴ Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science, and Station for Collaborative Research, Kyushu University, Maidashi, Fukuoka, 812-8582, Japan
⁵ Biochemistry Division, National Cancer Center Research Institute, 1-1 Tsukiji 5-chome, Chuo-ku, Tokyo, 104-0045, Japan
⁶ Laboratory of Molecular Human Genetics, Faculty of Pharmaceutical Sciences, Kanazawa University, 13-1 Takara-machi, Kanazawa, 920-0934, Japan

View larger version (42K): [in a new window]   Fig. 1. 14-3-3ß, 14-3-3, and 14-3-3 bind to CDC25B in transfected cells. HEK293 (left panels) or U2OS (right panels) cells were transfected with FLAG-tagged CDC25B together with either empty vector or one of the myc-tagged 14-3-3 subtypes as described in Materials and Methods. (Top row) Lysate. Expression of CDC25B and 14-3-3-subtypes was confirmed in cell lysates with anti-FLAG antibody against CDC25B or anti-myc antibody against 14-3-3, respectively. (Middle row) IP: CDC25B (-FLAG Ab). CDC25B was immunoprecipitated with anti-FLAG beads followed by western blotting and detection with anti-FLAG antibody to detect CDC25B and anti-myc antibody to detect CDC25B-bound 14-3-3. (Bottom row) IP: 14-3-3 (-myc Ab. Reciprocal immunoprecipitation; CDC25B was detected in anti-myc immunoprecipitates. Protein 14-3-3 subtypes were immunoprecipitated with anti-myc antibody; the collected 14-3-3 or 14-3-3-bound CDC25B was detected by immunoblotting. v, empty vector; ß, 14-3-3ß, ; 14-3-3, ; 14-3-3.

View larger version (34K): [in a new window]   Fig. 2. Binding of 14-3-3 subtypes to CDC25B is site specific. (A) Putative 14-3-3 consensus binding sites in CDC25B. (B-D) Mutants of CDC25B were transfected into HEK293 or U2OS cells either alone or together with 14-3-3 subtypes as indicated. Recovered CDC25B proteins are indicated (upper panel of each set of figures). The letters at the top and numbers at the bottom of each blot represent the CDC25B mutants: wild-type (1); S81A (2); S137A (3); S216A (4); S309A (5); S361A (6); 81S (7); 137S (8); 216S (9); 309S (10); 361S (11); 5SA (12). The definitions of the abbreviations for each mutant are described in the text. (B) Mutants of CDC25B were co-transfected into HEK293 cells with 14-3-3 subtypes ß, or . Protein expression was determined by immunoblot. Wild-type or mutant CDC25B proteins were immunoprecipitated with anti-FLAG beads, and CDC25B-bound 14-3-3 was determined in the lysate (Lysate) and the immunoprecipitate [IP: CDC25B (-FLAG Ab)]. Separate panel `long exposure' shows 14-3-3 subtype after an exposure for 1 hour. (C) Mutants of CDC25B were transfected into HEK293 cells. Recovered CDC25B proteins and CDC25B-bound endogenous 14-3-3ß (endo-14-3-3ß) or endogenous 14-3-3 (endo-14-3-3) were detected with specific antibodies in the lysate (Lysate) and the immunoprecipitate [IP: CDC25B (-FLAG Ab]. (D) Mutants of CDC25B were transfected into U2OS cells. Recovered CDC25B and CDC25B-bound endogenous 14-3-3 (endo-14-3-3) were detected with specific antibodies in the lysate (Lysate) and the immunoprecipitate [IP: CDC25B (-FLAG Ab].(E) Binding of endogenous and transfected 14-3-3 subtypes to CDC25B mutants. ++, well bound; +, detectably bound; ±, faintly bound (could be detected only after long exposure); -, no binding.

View larger version (48K): [in a new window]   Fig. 3. Efficient binding of 14-3-3 to CDC25B requires two independent sites. HEK293 cells were co-transfected with 14-3-3 and a series of CDC25B mutants. Protein expression (Lysate) and protein binding (IP: CDC25B (-FLAG Ab)) were detected. The letters in the upper panel of Lysate indicate CDC25B wild type and respective mutants. The definitions of the abbreviations for each mutant are described in the text.

View larger version (20K): [in a new window]   Fig. 4. 14-3-3ß but not 14-3-3 efficiently redistributes CDC25B from the nucleus to the cytoplasm. HEK293 cells were transfected with FLAG-tagged CDC25B in combination with empty vector, myc-tagged 14-3-3ß or myc-tagged 14-3-3, followed by immunostaining with anti-FLAG antibodies to detect the subcellular localization of CDC25B and with anti-myc antibodies to detect cotransfected 14-3-3 proteins. Analyses showed that more than 95% of the cells that expressed CDC25B also expressed the co-transfected 14-3-3 proteins. (A) Exemplary images, showing how the subcellular distribution of CDC25B was evaluated: N>C, predominantly nuclear; N=C diffuse; N<C, predominantly cytoplasmic. (B) Wild-type CDC25B was co-transfected with empty vector (WT), myc-tagged 14-3-3ß (WT+14-3-3ß) or myc-tagged 14-3-3 (WT+14-3-3) to quantify the subcellular distribution of CDC25B. Over 200 cells expressing CDC25B were counted to determine the percentage of cells that express CDC25B with nuclear, diffuse and cytoplasmic distribution. Error bars in graphs represent the means±s.d. of three independent experiments. (C) Transfection with wild-type CDC25B alone (WT), S309A mutant of CDC25B alone (S309A) and mutant S309A in combination with myc-tagged 14-3-3ß (S309A+14-3-3ß) or myc-tagged 14-3-3 (S309A+14-3-3). Over 200 cells expressing CDC25B were counted to determine the percentage of cells that express CDC25B with nuclear, diffuse and cytoplasmic distribution. Error bars in graphs represent the means±s.d. of three independent experiments. (D) Transfection with wild-type CDC25B alone (WT), S216A mutant of CDC25B alone (S216A) and mutant S216A in combination with myc-tagged 14-3-3ß (S216A+14-3-3ß) or myc-tagged 14-3-3 (S216A+14-3-3). Over 200 cells expressing CDC25B were counted to determine the percentage of cells that express CDC25B with nuclear, diffuse and cytoplasmic distribution. Error bars in graphs represent the means±s.d. of three independent experiments.

View larger version (38K): [in a new window]   Fig. 5. Only the 309S mutant of CDC25B was distributed diffusely with co-transfection of 14-3-3ß. (A) Wild type CDC25B or different CDC25B mutants with a single phosphorylatable serine were cotransfected with 14-3-3ß into HEK293 cells. (Upper panels) Subcellular localization of CDC25B wild type and mutants. (Lower panel) Corresponding images of nuclei. (B) Percentage of cells transfected with mutant CDC25B 309S (shown in A) that express CDC25B with nuclear, diffuse and cytoplasmic distribution. Over 200 cells expressing CDC25B were counted to determine the percentage of cells that express CDC25B. Transfection with wild-type CDC25B alone (WT), 309S mutant of CDC25B alone (309S) and mutant 309S in combination with myc-tagged 14-3-3ß (309S +14-3-3ß) or myc-tagged 14-3-3 (309S +14-3-3). Error bars in graphs represent the means±s.d. of three independent experiments. Subcellular distribution of CDC25B: N>C, predominantly nuclear; N=C diffuse; N<C, predominantly cytoplasmic (C).

View larger version (12K): [in a new window]   Fig. 6. 14-3-3 had effects similar to those of 14-3-3ß on the subcellular localization of CDC25B. HE293 cells were transfected with (A) wild-type CDC25B, (B) the 309S mutant or (C) the S309A mutant with or without 14-3-3. Over 200 cells expressing CDC25B were counted to determine the percentage of cells that express CDC25B with nuclear, diffuse and cytoplasmic distribution. Error bars in graphs represent the means±s.d. of three independent experiments. Subcellular distribution of CDC25B: N>C, predominantly nuclear; N=C diffuse; N<C, predominantly cytoplasmic (C).

View larger version (17K): [in a new window]   Fig. 7. Binding of 14-3-3ß to CDC25B efficiently slowed down the nuclear import of CDC25B induced by leptomycin B (LMB). (A) HEK293 cells transfected with wild-type CDC25B and treated with 20 ng/ml LMB at for 3 hours. Transfected CDC25B was detected with anti-FLAG antibody. The upper and lower panels show the results without or with LMB treatment, respectively. (B) HEK293 cells transfected with wild-type CDC25B alone or with 14-3-3ß. Twenty-four hours after transfection, 20 ng/ml LMB were added to the cultures and the percentage of cells exhibiting nuclear-specific localization of CDC25B was determined at the indicated time-points: 0 (before addition of LMB), 1.5, 3 or 6 hours after the addition. Over 200 cells expressing CDC25B were counted to determine the percentage of cells that express CDC25B. The percentage of cells with a nuclear localization (as shown in Fig. 4) was determined from three independent experiments. , CDC25B; ,CDC25B with 14-3-3ß.

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