RESUMO
Bone morphogenetic protein 7 (BMP-7) regulates cellular metabolism in embryonic and adult tissues. Signal transduction occurs through the activation of intracellular Smad proteins. In this paper, using a yeast two-hybrid screen, Smad1 was found to interact with the cytoplasmic domain of CD44, a receptor for the extracellular matrix macromolecule hyaluronan. Coimmunoprecipitation experiments confirmed the interaction of Smad1 with full-length CD44-interactions that did not occur when CD44 receptors truncated within the cytoplasmic domain were tested. Chondrocytes overexpressing a truncated CD44 on a background of endogenous full-length CD44 no longer exhibited Smad1 nuclear translocation upon BMP-7 stimulation. Further, pretreatment of chondrocytes with Streptomyces hyaluronidase to disrupt extracellular hyaluronan-cell interactions inhibited BMP-7-mediated Smad1 phosphorylation, nuclear translocation of Smad1 or Smad4, and SBE4-luciferase reporter activation. These results support a functional link between the BMP signaling cascade and CD44. Thus, changes in hyaluronan-cell interactions may serve as a means to modulate cellular responsiveness to BMP.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Cartilagem/metabolismo , Condrócitos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Matriz Extracelular/metabolismo , Receptores de Hialuronatos/metabolismo , Transdução de Sinais/genética , Transativadores/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Transporte Ativo do Núcleo Celular/efeitos dos fármacos , Transporte Ativo do Núcleo Celular/genética , Animais , Proteína Morfogenética Óssea 7 , Proteínas Morfogenéticas Ósseas/genética , Células COS , Cartilagem/citologia , Cartilagem/crescimento & desenvolvimento , Bovinos , Condrócitos/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Genes Reporter/genética , Humanos , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Hialuronoglucosaminidase/farmacologia , Mutagênese Sítio-Dirigida , Fosforilação/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteínas Smad , Proteína Smad1 , Proteína Smad4 , Transativadores/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transfecção , Fator de Crescimento Transformador beta/genética , Técnicas do Sistema de Duplo-HíbridoRESUMO
Polo-like kinase 3 (Plk3, alternatively termed Prk) is involved in the regulation of DNA damage checkpoint as well as in M-phase function. Plk3 physically interacts with p53 and phosphorylates this tumor suppressor protein on serine-20, suggesting that the role of Plk3 in cell cycle progression is mediated, at least in part, through direct regulation of p53. Here we show that Plk3 is rapidly activated by reactive oxygen species in normal diploid fibroblast cells (WI-38), correlating with a subsequent increase in p53 protein level. Plk3 physically interacts with Chk2 and the interaction is enhanced upon DNA damage. In addition, Chk2 immunoprecipitated from cell lysates of Daudi (which expressed little Plk3) is capable of stimulating the kinase activity of purified recombinant Plk3 in vitro, and this stimulation is more pronounced when Plk3 is supplemented with Chk2 immunoprecipitated from Daudi after DNA damage. Furthermore, ectopic expression Chk2 activates cellular Plk3. Together, our studies suggest Chk2 may mediate direct activation of Plk3 in response to genotoxic stresses.