Involvement of GSK-3beta and DYRK1B in differentiation-inducing factor-3-induced phosphorylation of cyclin D1 in HeLa cells.
Differentiation-inducing factors (DIFs) are putative morphogens that induce cell differentiation in Dictyostelium discoideum. We previously reported that DIF-3 activates glycogen synthase kinase-3beta (GSK-3beta), resulting in the degradation of cyclin D1 in HeLa cells. In this study, we investigated the effect of DIF-3 on cyclin D1 mutants (R29Q, L32A, T286A, T288A, and T286A/T288A) to clarify the precise mechanisms by which DIF-3 degrades cyclin D1 in HeLa cells. We revealed that T286A, T288A, and T286A/T288A 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-3beta and dual-specificity tyrosine phosphorylation regulated kinase 1B (DYRK1B) by RNA interference attenuated the DIF-3-induced cyclin D1 phosphorylation and degradation. The effect of DIF-3 on DYRK1B activity was examined and we found that DIF-3 also activated this kinase. Further, we found that not only GSK-3beta but also DYRK1B modulates cyclin D1 subcellular localization by the phosphorylation of Thr(288). These results suggest that DIF-3 induces degradation of cyclin D1 through the GSK-3beta- and DYRK1B-mediated threonine phosphorylation in HeLa cells.
Takahashi-Yanaga
Fumi
F
Department of Clinical Pharmacology, Faculty of Medical Sciences, Kyushu University, Fukuoka 812-8582, Japan. yanaga@clipharm.med.kyushu-u.ac.jp
Mori
Jun
J
Matsuzaki
Etsuko
E
Watanabe
Yutaka
Y
Hirata
Masato
M
Miwa
Yoshikazu
Y
Morimoto
Sachio
S
Sasaguri
Toshiyuki
T
D015153:Blotting, Western; D019938:Cyclin D1 / Q000378:metabolism*; D005434:Flow Cytometry; D038362:Glycogen Synthase Kinase 3 / Q000502:physiology*; D000071679:Glycogen Synthase Kinase 3 beta; D006367:HeLa Cells; D006588:Hexanones / Q000378:metabolism*; D006801:Humans; D006868:Hydrolysis; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D011505:Protein-Tyrosine Kinases / Q000502:physiology*; D034622:RNA Interference; D000097603:Dyrk Kinases
D019938:Cyclin D1; D038362:Glycogen Synthase Kinase 3; D000071679:Glycogen Synthase Kinase 3 beta; D006588:Hexanones; D017346:Protein Serine-Threonine Kinases; D011505:Protein-Tyrosine Kinases; D000097603:Dyrk Kinases; C055364:1-(3-chloro-2,6-dihydroxy-4-methoxyphenyl)-1-hexanone; C000605506:GSK3B protein, human