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.