A novel DYRK1B inhibitor AZ191 demonstrates that DYRK1B acts independently of GSK3β to phosphorylate cyclin D1 at Thr286, not Thr288

Abstract

DYRK1B (dual-specificity tyrosine phosphorylation-regulated kinase 1B) is amplified in certain cancers and may be an oncogene; however, our knowledge of DYRK1B has been limited by the lack of selective inhibitors. In the present study we describe AZ191, a potent small molecule inhibitor that selectively inhibits DYRK1B in vitro and in cells. CCND1 (cyclin D1), a key regulator of the mammalian G1-S-phase transition, is phosphorylated on Thr(286) by GSK3β (glycogen synthase kinase 3β) to promote its degradation. DYRK1B has also been proposed to promote CCND1 turnover, but was reported to phosphorylate Thr(288) rather than Thr(286). Using in vitro kinase assays, phospho-specific immunoblot analysis and MS in conjunction with AZ191 we now show that DYRK1B phosphorylates CCND1 at Thr(286), not Thr(288), in vitro and in cells. In HEK (human embryonic kidney)-293 and PANC-1 cells (which exhibit DYRK1B amplification) DYRK1B drives Thr(286) phosphorylation and proteasome-dependent turnover of CCND1 and this is abolished by AZ191 or DYRK1B RNAi, but not by GSK3β inhibitors or GSK3β RNAi. DYRK1B expression causes a G1-phase cell-cycle arrest, but overexpression of CCND1 (wild-type or T286A) fails to overcome this; indeed, DYRK1B also promotes the expression of p21CIP1 (21 kDa CDK-interacting protein 1) and p27KIP1 (CDK-inhibitory protein 1). The results of the present study demonstrate for the first time that DYRK1B is a novel Thr(286)-CCND1 kinase that acts independently of GSK3β to promote CCND1 degradation. Furthermore, we anticipate that AZ191 may prove useful in defining further substrates and biological functions of DYRK1B.

The Down syndrome-related protein kinase DYRK1A phosphorylates p27Kip1and Cyclin D1 and induces cell cycle exit and neuronal differentiation

“…Two recent reports have now made clear that either DYRK1A or DYRK1B can promote Cyclin D1 degradation via direct phosphorylation of Thr286 but do not target Thr288 16 , 41 . Consistent with our results for the effect of DYRK1A in SH-SY5Y cells, Ashford et al 16 have found in the pancreatic carcinoma cell line PANC-1 that the DYRK1B-driven phosphorylation at Thr286 and turnover of Cyclin D1 were not reduced by GSK3β inhibition. Thus, DYRK1A and DYRK1B regulate Cyclin D1 stability independent of GSK3β activity at least in some cell types.…”

“…Although GSK3β was initially reported to phosphorylate Cyclin D1 on Thr286, 68 DYRK1B was later proposed to promote Cyclin D1 degradation by phosphorylation at Thr288 69 . Two recent reports have now made clear that either DYRK1A or DYRK1B can promote Cyclin D1 degradation via direct phosphorylation of Thr286 but do not target Thr288 16 , 41 . Consistent with our results for the effect of DYRK1A in SH-SY5Y cells, Ashford et al 16 have found in the pancreatic carcinoma cell line PANC-1 that the DYRK1B-driven phosphorylation at Thr286 and turnover of Cyclin D1 were not reduced by GSK3β inhibition.…”

“…Several kinases have been proposed to target this residue, including KIS (kinase interacting with stathmin), AKT, ERK2, CDK5, HIPK2 (homeodomain-interacting protein kinase 2), and DYRK1B 9 , 11 - 15 . Proteasomal degradation of Cyclin D1 during G 1 phase is induced by phosphorylation at Thr286 via GSK3β 10 , but recently DYRK1A and DYRK1B were also shown to phosphorylate this residue in non-neural cells 16 . The phosphorylation of p27 Kip1 and Cyclin D1 has mainly been investigated in cancer cell cycle progression, and the respective regulative kinases in neuronal progenitor cells remain uncharacterized.…”

Bafilomycin A1 induces caspase-independent cell death in hepatocellular carcinoma cells via targeting of autophagy and MAPK pathways

“…2D), suggesting that GSK3β may not be implicated in BafA1-induced Cyclin D1 turnover in HCC cells. Previous studies, in addition to more recent work in our laboratory have shown that DYRK1B (dual-specificity tyrosine phosphorylation-regulated kinase 1B) promotes the turnover of Cyclin D1 in a GSK3β-independent manner1112. A steady-state increase in DYRK1B protein levels was observed in both BEL7402 and HepG2 cells following treatment with BafA1 (Fig.…”

Degrons in cancer

“…Cyclin D1 is a promoter of the G 1 -S transition in cell cycle and contains a phosphodegron recognized by SCF E3 ligase with the FBXO4 or the CRL7 E3 ligase with the FBXW8 receptor subunits (223)(224)(225). Cyclin D1 functions in the G 1 phase when bound to CDK4.…”