Previously, we reported that (S)-3,5-dihydroxypenylglycine (DHPG), an agonist for group I metabotropic glutamate receptors (mGluRs), stimulates CK1 and Cdk5 kinase activities in neostriatal neurons, leading to enhanced phosphorylation, respectively, of Ser-137 and Thr-75 of DARPP-32 (dopamine and cAMP-regulated phosphoprotein, 32 kDa). We have now investigated the signaling pathway that leads from mGluRs to casein kinase 1 (CK1) activation. In mouse neostriatal slices, the effect of DHPG on phosphorylation of Ser-137 or Thr-75 of DARPP-32 was blocked by the phospholipase C inhibitor U73122, the Ca 2؉ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid (BAPTA/ AM), and the calcineurin inhibitor cyclosporin A. In neuroblastoma N2a cells, the effect of DHPG on the activity of transfected HA-tagged CK1⑀ was blocked by BAPTA/AM and cyclosporin A. In neostriatal slices, the effect of DHPG on Cdk5 activity was also abolished by BAPTA/AM and cyclosporin A, presumably through blocking activation of CK1. Metabolic labeling studies and phosphopeptide mapping revealed that a set of C-terminal sites in HA-CK1⑀ were transiently dephosphorylated in N2a cells upon treatment with DHPG, and this was blocked by cyclosporin A. A mutant CK1⑀ with a nonphosphorylatable C-terminal domain was not activated by DHPG. Together, these studies suggest that DHPG activates CK1⑀ via Ca 2؉ -dependent stimulation of calcineurin and subsequent dephosphorylation of inhibitory C-terminal autophosphorylation sites.
Casein kinase 1 (CK1)1 was one of the first serine/threonine protein kinases to be isolated and characterized. There are at least seven mammalian CK1 isoforms (␣, , ␥1, ␥2, ␥3, ␦, and ⑀ (1, 2)), and distinct CK1 family members are likely to have a variety of roles in eukaryotic cells. An increasing number of potential physiologic substrates for CK1 isoforms have been identified. CK1␣ phosphorylates M1 and M3 muscarinic receptors and rhodopsin in an agonist-dependent manner (3, 4). CK1⑀ and CK1␦ phosphorylate N-terminal residues of p53 in vitro and in vivo, and DNA-damaging drugs enhance this activity (4 -6). CKI⑀ is an important regulator of -catenin in the Wnt pathway; CKI⑀ mimicked Wnt in inducing a secondary axis in Xenopus, stabilizing -catenin, and stimulating -catenin-dependent gene transcription (7-11). In Drosophila, the double-time gene product, a CK1⑀ homolog, has been found to interact with dPER and regulate circadian cycle length (12). CK1␦ and CK1⑀ have also both been implicated in the regulation of the circadian clock in mammals (13-15).CK1 family members contain a highly related, central kinase domain that is flanked by N-and C-terminal extensions of variable length. The amino acid sequences of the C-terminal extensions are in general not highly related. However, the 124-amino acid C-terminal domain of mammalian CK1⑀ is 50% identical to that of CK1␦. Notably, several in vitro studies have shown that the activities of CK1␦ and CK1⑀ are regulated by autophosphorylation of their respective C-terminal domains (...