CONTROLE DO CICLO CELULAR DEGRADATION OF Cdc25A BY β-TrCP DURING S PHASE AND IN RESPONSE TO DNA DAMAGE BUSINE, L. et al. Nature, v. 426, n. 6, november 2003, p. 87-91 HENRIQUE FABIANO DO NASCIMENTO
Introdução: ► Cdc25A fosfatase é essencial para a progressão do ciclo celular por causa de sua função de desfosforilar cdks (B/cdc2). ► Em resposta a danos no DNA ou falência da replicação, as ATM e ATR proteínas cinases ativam as checkpoint cinases Chk1 e Chk2 que levam a hiperfosforilação do Cdc25A. Estes eventos estimulam a proteólise da Cdc25A mediada por ubiquitina e contribuem para o atraso do ciclo celular, prevenindo a instabilidade genômica. ► β-TrCP é uma proteína F-box que fosforila Cdc25A para degradação pelo complexo protéico Skp1/Cul1/F-box (SCF). ► Downregulation da expressão de β-TrCP1 e β-TrCP2 causa um acúmulo de Cdc25A nas células em fase S e previne a degradação de Cdc25A induzida por radiação ionizante, indicando que β-TrCPpode funcionar em checkpoint dentro da fase S.
B B cdc2 cdc2 p KEN BOX cdc25 SC Fator promotor de anáfase (APC) ou ciclossomo Mitose KEN BOX cdc25 ubiquitina SC p F-box (SCF) degradação pós-mitose (senão como formar MPF “inativo” para o outro ciclo?) SC KEN BOX ubiquitina resiste a APC, mas é lábil e degradada em resposta a UV (checkpoint, mas qual mecanismo então??)
Metodologia ► Culturas de células, sincronização e transfecção; ► Plasmídios (Flag- e His- tagged Cdc25A mutantes); ► Imunoblotting, imunopreciptação e tratamento com fosfatase; ► Ensaio de ligação com peptídeo; ► Ubiquitinação in vivo; ► siRNA; ► Ensaio de síntese de DNA radio-resistente.
Resultados: Únicas que interagem com Cdc25A in vivo • alguma F (do SCF) se liga in vivo a cdc25? Qual?? • Céls HeLa + F com “flag” • Imunoprecip com anti-flag • WB revelado com anti-cdc25 • ou com anti-C, ou anti-flag (controle) Figure 1 Cdc25A interacts with -TrCP1 and -TrCP2 in vivo. a, HeLa cells were transfected with the indicated Flag–tagged F-box protein constructs. F-box proteins were immunoprecipitated from extracts with an anti-Flag resin and immunocomplexes were blotted with antibodies specific for Cul1, Cdc25A and Flag. b, HeLa cells were co-transfected with a vector expressing His–Cdc25A plus the indicated Flag-tagged F-box protein constructs. Immunocomplexes were analysed as in a. WCE, whole-cell extract. Asterisk indicates the position of the IgG heavy chain (-TrCP2 protein overlaps with IgG). c, Cdc25A bound to -TrCP proteins is phosphorylated. Immunocomplexes obtained by -TrCP1 immunoprecipitation were treated with -phosphatase and analysed for Cdc25A. • Céls HeLa + cdc25a com flag (6 his) • Imunoprecip com anti-his • WB revelado com anti Fs • OPS: cdc25 fosforilada! (IP anti-F) hiperfosforilação
► A sequência de aminoácidos de Cdc25A contém um motivo DSGXXXXS (DSG(X)4S) ►Que é similar com os motivos DSGXXS ou DSGXXXS presentes em conhecidos substratos do complexo SCF β-TrCP . ► Fosforilação em ambas serinas é necessária para a ligação com β-TrCP e consequentq degradação. ► Substituição de duas serinas por alaminas abole a interação deCdc25A com β-TrCP . Esta interação é provavelmente dependente da fosforilação dos resíduos de serina (fig. 2b).
Resultados: Figure 2 Interaction with -TrCP protein through a phosphorylated DSG motif is required for Cdc25A degradation and polyubiquitination. a, Alignment of DSG motifs identified in known -TrCP substrates. Serine-to-alanine mutations in the DSG motif of Cdc25A are boxed. b, Vectors expressing His-tagged Cdc25ADSG2 and Cdc25ADSG3 were coexpressed with Flag–-TrCP1 in HeLa cells, and anti-Flag immunocomplexes were blotted for Cdc25A. c, Immobilized Cdc25A-derived peptides, with or without phosphorylation on Ser 82 and Ser 88 (a), were incubated with [35S]methionine-labelled in vitro translated F-box proteins (IVT) and analysed by autoradiography. d, HeLa cells were transfected with the indicated Flag-tagged constructs and analysed for Cdc25A expression after cycloheximide (CHX) treatment. e, [35S]methionine-labelled in vitro translated Cdc25A was incubated with HeLa cell extract enriched with the indicated Skp1–F-box protein complexes and analysed by autoradiography. f, The ATP analogue AMP-PNP was used in the ubiquitin ligation reaction. g, -TrCP-mediated ubiquitination of wild-type Cdc25A and the Cdc25ADSG3 mutant. Mutantes são mais estáveis Ubiquitinação Fosforilação é requerida p/ a degradação
Resultados: Cdc25 Ken = mutante que não é degradado pelo complexo APC/C G1/S Figure 3 -TrCP controls Cdc25A abundance during progression of S phase. a, HeLa cells were mock-transfected or transfected with -TrCP1 and -TrCP2 siRNA oligonucleotides. Expression of Cdc25A and -TrCP1/2 (analysed by immunoblotting and quantitative PCR) is shown. b, Cells transfected as indicated were synchronized by double-thymidine block and released in nocodazole-containing medium. Cells were collected at the indicated time points, lysed and immunoblotted for Cdc25A, Emi1 and cyclin A. Samples collected at the T0, T6 an T12 time points were aligned for a direct comparison of Cdc25A expression. c, Cells transfected as indicated were synchronized by nocodazole treatment and released in drug-free medium. Cells were analysed for Cdc25A, Emi1 and cyclin B1 as in b. d, Cells transfected with a vector expressing Flag-tagged wild-type Cdc25A or a Cdc25AKEN2 mutant were subjected to RNA interference and analysed for overexpression of Cdc25A. M/G1
Resultados: Figure 4 -TrCP is required for degradation of Cdc25A induced by ionizing radiation in the intra-S-phase checkpoint. a, HeLa cells were mock-transfected or transfected with -TrCP or Cdh1 siRNA and after 48 h were exposed to ionizing radiation (10 Gy). Cdc25A protein is shown at low and high exposure. b, siRNA-transfected S-phase cells were exposed to ionizing radiation, and the half-life of Cdc25A was analysed by cycloheximide (CHX) treatment. c, Percentage of DNA synthesis, normalized against mock-transfected non-irradiated cells, was assessed in mock-transfected cells and in cells transfected with -TrCP1/2 or -TrCP1/2 plus Cdc25A siRNA, 90 min after ionizing radiation treatment. d, HeLa cells overexpressing Flag–Cdc25A were irradiated with 10 and 20 Gy in the presence of the proteasome inhibitor MG132 and collected at the indicated time points after irradiation. Immunoprecipitated Cdc25A was immunoblotted with a purified anti-phosphoS82/S88 antibody. e, U2OS cells stably expressing wild-type Cdc25A or Cdc25ADSG2 were mock-treated or treated by ionizing radiation. f, Flag-tagged Cdc25A immunocomplexes were immunoblotted for Cdc25A, Cul1, Skp1 and -TrCP1. Mutante: pouca degradação após raio X
Conclusões: ►É requerido dois sites de fosforilação no Cdc25A para sua degradação por β-TrCP; ►Fosforilação nos resíduos de Serina pode estimular a degradação de Cdc25A por facilitar a interação com componentes do SCF ou por aumentar a habilidade do SCF para catalisar uma poliubiquitinação.