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

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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.