A novel phosphorylation motif for casein kinase 1 (CK1) in response to two sulfated lipids [sulfatide and cholesterol-3-sulfate (SCS)] was determined, using three functional proteins [myelin basic protein (MBP), tau protein (TP) and RhoA (a small GTPase)] and five synthetic MBP peptides as phosphate acceptors for the kinase in vitro. It was found that (i) MBP, p8 (positions 38-118) cleaved from MBP, and a synthetic peptide M103 were effectively phosphorylated by CK1delta in the presence of SCS; (ii) sulfatide in comparison with CH-3S highly enhanced autophosphorylation of CK1delta; (iii) SCS had a high binding affinity with MBP and peptide M103, but not other MBP peptides lacking K-G-R; and (iv) a novel consensus phosphorylation motif (K/R-X-K/R-X-X-S/T) for CK1 was identified among several SCS-binding proteins (SCS-BPs) and three CK1 isoforms (delta, epsilon and gamma). The binding of SCS to two basic brain proteins (MBP and TP) resulted in the high stimulation of their phosphorylation by three CK1 isoforms (alpha, delta and epsilon), but not CK1gamma. In contrast, an acidic protein (RhoA) was effectively phosphorylated by CK1delta in the presence of SCS, and also highly phosphorylated by CK1gamma in the presence of sulfatide. Our results presented here suggest that (i) sulfatide may function as an effective stimulator for autophosphorylation of CK1; and (ii) cellular SCS-binding proteins, containing novel phosphorylation motifs for CK1, may be preferentially phosphorylated by CK1 with isoform specificity at the highly accumulated level of SCS in the brain.