D4476, a cell-permeant inhibitor of CK1, suppresses the site-specific phosphorylation and nuclear exclusion of FOXO1a

Abstract

The protein kinase CK1 phosphorylates serine residues that are located close to another phosphoserine in the consensus pSer-Xaa-Xaa-Ser. This specificity generates regions in its target proteins containing two or more neighbouring phosphoserine residues, termed here multisite phosphorylation domains (MPDs). In this paper, we demonstrate that D4476 is a potent and rather selective inhibitor of CK1 in vitro and in cells. In H4IIE hepatoma cells, D4476 specifically inhibits the phosphorylation of endogenous forkhead box transcription factor O1a (FOXO1a) on Ser322 and Ser325 within its MPD, without affecting the phosphorylation of other sites. Our results indicate that these residues are targeted by CK1 in vivo and that the CK1-mediated phosphorylation of the MPD is required for accelerated nuclear exclusion of FOXO1a in response to IGF-1 and insulin. D4476 is much more potent and specific than IC261 or CKI-7, and is therefore the most useful CK1 inhibitor currently available for identifying physiological substrates of CK1.

Figure 1

Structures of CK1 inhibitors and their effects on CK1 activity in vitro. (A) Structures of D4476 and IC261 (Mashhoon et al, 2000). (B) Purified CK1δ was assayed with phosphorylated peptide TFRPRTSpSNASTIS (30 μM), corresponding to the sequence surrounding residues 312–325 of FOXO1a, at an ATP concentration of 0.1 mM and varying concentrations of D4476 (filled circles), IC261 (open squares) and CKI-7 (open circles). The results are the average of at least four separate determinations, each performed in duplicate.

Figure 2

D4476 inhibits the phosphorylation of FOXO1a at Ser322 and Ser325, but not at Thr24 in vitro. Bacterially expressed GST-FOXO1a (1 μM) was left unphosphorylated (−) or maximally phosphorylated for 30 min at 30°C with 1 U ml⁻¹ PKB, 10 mM magnesium acetate and 0.1 mM ATP, followed by phosphorylation for 10 min with 30 mU ml⁻¹ CK1 (+), in the presence of the indicated concentrations of D4476. Aliquots of the reaction were spotted onto nitrocellulose and immunoblotted with phospho-specific antibodies recognizing phosphorylated Thr24 (pThr24), Ser322 (pSer322) and Ser325 (pSer325) and an antibody that recognizes the phosphorylated and unphosphorylated forms of FOXO1a equally well (FOXO1a). Similar results were obtained in several independent experiments.

Figure 3

D4476 specifically inhibits the phosphorylation at Ser322 and Ser325 specifically in H4IIE cells. Cells were serum starved for 4 h, and then stimulated for 30 min without (−) or with (+) 20 nM insulin. Aliquots of the cell lysates (2.5 mg protein) were then immunoprecipitated with 20 μg of anti-FOXO1a antibody raised against the whole protein. After washing the immunoprecipitates and denaturation in SDS, aliquots of the solubilized material were subjected to SDS–polyacrylamide gel electrophoresis. Following transfer to nitrocellulose, the membranes were subjected to immunoblotting using the antibodies used in Fig 1, as well as with a phospho-specific antibody that recognizes Ser329 (pSer329). Where indicated, the cells were incubated for 60 min with various concentrations of D4476 prior to stimulation with insulin. Similar results were obtained in at least six independent experiments.

Figure 4

D4476 inhibits IGF-1 and serum-stimulated nuclear exclusion of FOXO1a in living cells. HEK293 cells were transfected with wild-type FOXO1a–GFP or mutant (Ser319Ala) FOXO1a–GFP. At 10 h post-transfection, the cells were serum starved for 12 h, and then stimulated in the presence of 50 ng ml⁻¹ IGF-1 and 10% fetal calf serum with or without 150 μM D4476 pretreatment (10 min). Initial rates of nuclear exclusion were determined as described previously (Rena et al, 2002) by time-lapse imaging of live cells using confocal microscopy on a heated stage. Closed circles indicate the initial nuclear exclusion rate of FOXO1a in the absence of D4476. Open circles indicate the initial nuclear exclusion rate of FOXO1a in the presence of D4476. Squares indicate the initial nuclear exclusion rate of Ser319Ala FOXO1a–GFP. Each point is the average of seven cells.