A Novel Consensus Phosphorylation Motif in Sulfatide- and Cholesterol-3-sulfate-Binding Protein Substrates for CK1 in Vitro

Abstract

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.

Biochemical Characterization of Epigallocatechin-3-gallate as an Effective Stimulator for the Phosphorylation of Its Binding Proteins by Glycogen Synthase Kinase-3&beta; <i>in Vitro</i>

“…Our two observations that (i) EGCG at low doses (1- P-Labeled GSK-3b in the reaction mixtures was determined by the membrane filter method, 11) as described in the Materials and Methods. Lineweaver-Burk plots of ATP for GSK-3b in the absence (᭺) or presence of 0.3 mM EGCG (᭹).…”

“…After drying, the 32 P-radioactivity on the membrane filter was measured with a liquid scintillation spectrophotometer, as described in our previous reports. 11,23) Phosphopeptide Mapping Briefly, either bMBP or hrTP (approx. 2 mg) in reaction mixtures (50 ml) comprising 40 mM Tris-HCl (pH 7.6), 2 mM DTT, 3 mM Mn 2ϩ and 5 mM [g-32 P]ATP (500 cpm/pmol) in the absence or presence of these two substrate proteins were phosphorylated for 240 min at 30°C by GSK-3b (approx.…”

“…Separated 32 P-labeled polypeptides cleaved from bMBP or hrTP were detected by autoradiography, as described in our previous reports. 11,23) Measurement of the Binding Affinities of EGCG with Four Substrate Proteins by a Quarts Crystal Microbalance (QCM) The binding affinities of four substrate proteins (bMBP, hrTP, rCRMP-2 and hrVM) with EGCG and its related two compounds (luteolin and quercetin) were separately determined by using a QCM (Initium, Tokyo, Japan) using bovine serum albumin (BSA) as an negative control, as described in our previous reports. 11,23) …”

“…11) A number of epidemiological studies have shown that the consumption of green tea may account for the anti-oxdant, anti-inflammatory, anti-carcinogenic, and neuropotective effects.…”

“…Although it has been reported that GSK-3b is a protein kinase responsible for hyperphosphorylation of TP in an early stage of Alzheimer's disease (AD), 25) one of human neurodegenerative disorder diseases, the EGCG-induced high phosphorylation of TP by GSK-3b is different from the GSK-3b-mediated hyperphosphorylation of TP primed by other Ser/Thr-protein kinases, such as CK1 and A-kinase and C-kinase, which are sensitive to EGCG in vitro. 11,26) Previously, we reported that both PI and SH can function as effective stimulators for the GSK-3b-mediated phosphorylation of bMBP through their selective binding to the Arg/Lys-rich domains, containing the consensus phosphorylation sites (Ser-residues) for the kinase, on the basic brain protein.…”