Studies on the signal transduction pathway of differentiation inducing factor to induce cyclin D1 degradation

分化诱导因子(DIF)是盘基网柄菌中诱导细胞分化的一种可能的形态因子。我们报道了DIF-3激活糖原合成酶β(GSK-3β)，导致细胞周期蛋白D1的降解。在本研究中，我们研究了DIF-3对细胞周期蛋白D1突变体(Arg29Gln、Leu32Ala、Thr286Ala、Thr288Ala和Thr286/288Ala)的影响，以阐明DIF-3在HeLa细胞中降解细胞周期蛋白D1的确切机制。与野生型细胞周期蛋白D1相比，Thr286Ala、Thr288Ala和Thr286/288Ala突变体对DIF-3诱导的细胞周期蛋白D1降解具有抗性，说明Thr^&lt；286&gt；和Thr^&lt；288&gt；在DIF-3诱导细胞周期蛋白D1降解过程中起关键作用。的确，DIF-3显著提高了细胞周期蛋白D1的磷酸化水平，而Thr^-lt；286&gt；和/或Thr^&lt；288&gt；的突变阻止了DIF-3诱导的磷酸化。去除内源性β和双特异性酪氨酸磷酸化调节蛋白1B可减弱DIF-3诱导的细胞周期蛋白D1的磷酸化和降解。这些结果表明，DIF-3通过β和DYRK1B介导的苏氨酸磷酸化诱导细胞周期蛋白D1的降解。

Differentiation-inducing factors (DIFs) are putative morphogens that induce cell differentiation in Dictyostelium discoideum. We reported that DIF-3 activates glycogen synthase kinase-3β (GSK-3β), resulting in the degradation of cyclin D1 in HeLa cells. In this study, we investigated the effect of DIF-3 on cyclin D1 mutants (Arg29Gln, Leu32Ala, Thr286Ala, Thr288Ala and Thr286/288Ala) to clarify the precise mechanisms by which DIF-3 degrades cyclin D1 in HeLa cells. We revealed that Thr286Ala, Thr288Ala, and Thr286/288Ala mutants were resistant to DIF-3-induced degradation compared with wild-type cyclin D1, indicating that the phosphorylation of Thr^<286> and Thr^<288> were critical for cyclin D1 degradation induced by DIF-3. Indeed, DIF-3 markedly elevated the phosphorylation level of cyclin D1, and mutations introduced to Thr^<286> and/or Thr^<288> prevented the phosphorylation induced by DIF-3. Depletion of endogenous GSK-3β and dual-specificity tyrosine-phosphorylation regulated kinase 1B (DYRK1B) by RNA interference attenuated the DIF-3-induced cyclin D1 phosphorylation and degradation. These results suggest that DIF-3 induces degradation of cyclin D1 through the GSK-3β- and DYRK1B-mediated threonine phosphorylation.