Casein kinase I (CK-I) is a ubiquitous and highly conserved second messenger-independent serine (Ser)/threonine (Thr)-protein kinase with a molecular weight of 25-55 kDa.1) Recently, we reported that (i) CK-I phosphorylates preferentially Thr-residues on high mobility group protein 1 (HMG1) in the presence of cholesterol-3-sulfate (CH-3S), a metabolite of cholesterol formed during squamous cell differentiation; (ii) this phosphorylation is selectively inhibited by heparin, but significantly stimulated by 3Ј,4Ј,7-trihydroxyisoflavone at low doses (0.1-3 mM); and (iii) CH-3S directly induces drastic conformational changes of HMG1.2) From these results, we conclude that HMG1 is a CH-3S-binding protein and CH-3S acts as an effective activator for the phosphorylation of HMG1 by CK-I in vitro. 2,3)An isoform h of C-kinase (C-kinase h, approx. 80 kDa) is classified as a Ca 2ϩ -independent and phospholipid-dependent protein kinase. 1,4) This isoform has a unique tissue distribution and is expressed predominantly in epithelial tissues, including skin, bone, esophagus, stomach, intestine, trachea and bronchus. [5][6][7] In particular, C-kinase h in the skin is localized exclusively in the outermost granular layer, which consists of terminally differentiating keratinocytes containing keratohyalin granules. 8) Rough endoplasmic reticulum is the site of intercellular localization of C-kinase h. 9) Furthermore, it has been reported that (i) CH-3S effectively activates C-kinase h rather than the other isoforms (C-kinase e and C-kinase d) in vitro; (ii) sulfatide (cerebroside sulfate) also activates C-kinase h to a lesser extent than CH-3S; (iii) the incubation of C-kinase h with ATP in the presence of CH-3S or both phosphatidylserine (PS) and phorbol-12,13-dibutyrate (PDBu) results in a significant induction of its autophosphorylation; and (iv) the activated C-kinase h efficiently phosphorylates myelin basic protein (MBP) and protamine sulfate in vitro. [10][11][12] In addition, Vila et al. have been reported that CK-I significantly phosphorylates C-kinase in the presence of Ca 2ϩ and phospholipids in vitro. 13) However, the phosphorylation of the specific C-kinase isoforms by CK-I and their physiological significance in vitro remain to be elucidated. Therefore, to clarify the physiological correlation between rhC-kinase h (a CH-3S-binding protein kinase) and CK-I in vitro, we set out to (i) detect the phosphorylation of five recombinant C-kinase isoforms (a, bII, g, d and h) by CK-I and the stimulatory effect of CH-3S on the CK-I-mediated phosphorylation of rhC-kinase h; and (ii) determine the physiological significance of the CK-I-mediated phosphorylation of rhC-kinase h in the presence of CH-3S. Phosphorylation of C-Kinase h h by CK-I in Vitro Phosphorylation of C-kinase h by CK-I was measured in standard reaction mixtures (50 ml) comprising 40 mM Tris-HCl (pH 7.6), 2 mM DTT, 3 mM Mn 2ϩ , rhC-kinase h (approx. 50 ng), CK-I (approx. 20 ng), 5 mM [g-32 P]ATP (500 cpm/pmol) and 3 mM CH-3S. After incubation for the indicated pe...

The inhibitory effects of (-)-matairesinol (MTS) isolated from Thujopsis dolabrata var. hondai on the activities of four distinct Ser/Thr-protein kinases [two casein kinases (CK-I and CK-II), A-kinase and C-kinase] were determined in vitro. It was found that (i) MTS inhibits the activities of CK-I and C-kinase alpha (ID(50)=approx. 10 microM) in a dose-depedent manner, but high doses are required to inhibit A-kinase activity (ID(50)=approx. 90 microM); (ii) the autophosphorylation of CK-I is more sensitive to MTS (ID(50)=approx. 0.2 microM); (iii) MTS inhibits CK-I activity in a manner similar to that observed with CK-I-7 (a CK-I inhibitor); and (iv) the compound inhibits CK-I activity by affecting ATP binding in a mixed type manner. These results indicate that MTS is a potent CK-I inhibitor in vitro.

The physiological correlation between casein kinase 2 (CK2) and two Src family tyrosine kinases (Src-TKs, Fyn and Src) was mainly investigated in vitro. It was found that (i) Thr-residues of these two Src-TKs were preferentially phosphorylated by CK2 using [g g-32 P]GTP as a phosphate donor; (ii) this phosphorylation was highly stimulated in the presence of poly-Arg; (iii) full phosphorylation of two Src-TKs by CK2 resulted in significant reduction of their TK activities; and (iv) quercetin (a CK2 inhibitor) inhibited the CK2-mediated reduction of their Src-TK activities in vitro. Under the same experimental conditions, similar results were obtained with Yes. These results suggest that CK2 may be a protein kinase responsible for the suppression of at least three Src-TKs (Fyn, Src and Yes) through the specific phosphorylation of their Thr-residues at the cellular level.