Abstract
The cyclic AMP response element-binding protein (CREB) initiates transcriptional responses to a wide variety of stimuli. CREB activation involves its phosphorylation on Ser-133, which promotes interaction between the CREB kinase-inducible domain (KID) and the KID-interacting domain of the transcriptional coactivator, CREB-binding protein (CBP). The KID also contains a highly conserved phosphorylation cluster, termed the ATM/CK cluster, which is processively phosphorylated in response to DNA damage by the coordinated actions of ataxia-telangiectasia-mutated (ATM) and casein kinases (CKs) 1 and 2. The ATM/CK cluster phosphorylation attenuates CBP binding and CREB transcriptional activity. Paradoxically, it was recently reported that DNA damage activates CREB through homeodomain-interacting protein kinase 2-dependent phosphorylation of Ser-271 near the CREB bZIP DNA binding domain. In this study we sought to further clarify DNA damage-dependent CREB phosphorylation as well as to explore the possibility that the ATM/CK cluster and Ser-271 synergistically or antagonistically modulate CREB activity. We show that, rather than being induced by DNA damage, Ser-270 and Ser-271 of CREB cophosphorylated in a CDK1-dependent manner during G2/M phase. Functionally, we show that phosphorylation of CREB on Ser-270/Ser-271 during mitosis correlated with reduced CREB chromatin occupancy. Furthermore, CDK1-dependent phosphorylation of CREB in vitro inhibited its DNA binding activity. The combined results suggest that CDK1-dependent phosphorylation of CREB on Ser-270/Ser-271 facilitates its dissociation from chromatin during mitosis by reducing its intrinsic DNA binding potential.
Highlights
CAMP response element-binding protein (CREB) is a transcriptional regulator that undergoes complex phosphoregulation in response many physiologic stimuli
During the course of cell cycle synchronization experiments we found that the antimitotic drugs nocodazole and colcemid induced a cAMP response element-binding protein (CREB) electrophoretic mobility shift; the magnitude of this shift exceeded that caused by ␥-irradiation or exposure to the DNA-damaging agent, etoposide (Fig. 1A, isoform 3). -Phosphatase treatment of extracts prepared from nocodazoletreated cells abolished the CREB electrophoretic mobility shift, Noc Col Eto IR forskolin and isobutylmethylxanthine (FI)
In this study we have demonstrated that CREB is phosphorylated at Ser-270/Ser-271 in a Cyclin-dependent kinase 1 (CDK1)-dependent manner during mitosis
Summary
Cell Culture and Drug Treatment—HeLa and HEK 293T cells were obtained from American Type Culture Collection and maintained in Dulbecco’s modified Eagle’s medium with 10% fetal bovine serum and 1% penicillin-streptomycin. Plasmids and siRNA—The expression construct pcDNA3.1Zeo encoding the 341-amino acid CREB protein with an aminoterminal FLAG tag was previously generated by our laboratory [25]. Whole cell extracts were prepared by incubating cells in high salt lysis buffer (25 mM HEPES, pH 7.4, 300 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 0.1% Triton X-100) supplemented with protease and phosphatase inhibitors for 10 min on ice as described previously by Shanware et al [25]. For unlabeled in vitro immune complex kinase assays, control IgG or CDK1-B1 protein complex was incubated with 0.5 g of His-CREBWT or His-CREBS270A/S271A in the presence of 100 M ATP as described above. Fixed cells were incubated with primary antibodies specific for CREB (see above) diluted in TBS-T containing 3% bovine serum albumin. Images were gathered using Carl Zeiss Axiovert 200 inverted fluorescence microscope as previously described [40]
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