Tokumitsu H et al. (1999) J Biol Chem "Substrate recognition by Ca2+/Calmodulin-dependent protein kinase kinase. Role of the arg-pro-rich insert domain."

Takahashi N, Yano S, Eto K, Muramatsu M, Tokumitsu H, Soderling TR

[J Biol Chem, 1999]

Mammalian Ca2+/CaM-dependent protein kinase kinase (CaM-KK) has been identified and cloned as an activator for two kinases, CaM kinase I (CaM-KI) and CaM kinase IV (CaM-KIV), and a recent report (Yano, S., Tokumitsu, H., and Soderling, T. R. (1998) Nature 396, 584-587) demonstrates that CaM-KK can also activate and phosphorylate protein kinase B (PKB). In this study, we identify a CaM-KK from Caenorhabditis elegans, and comparison of its sequence with the mammalian CaM-KK alpha and beta shows a unique Arg-Pro (RP)-rich insert in their catalytic domains relative to other protein kinases. Deletion of the RP-domain resulted in complete loss of CaM-KIV activation activity and physical interaction of CaM-KK with glutathione S-transferase-CaM-KIV (T196A). However, CaM-KK autophosphorylation and phosphorylation of a synthetic peptide substrate were normal in the RP-domain mutant. Site-directed mutagenesis of three conserved Arg in the RP- domain of CaM-KK confirmed that these positive charges are important for CaM-KIV activation. The RP- domain deletion mutant also failed to fully activate and phosphorylate CaM-KI, but this mutant was indistinguishable from wild-type CaM-KK for the phosphorylation and activation of PKB. These results indicate that the RP-domain in CaM-KK is critical for recognition of downstream CaM-kinases but not for its catalytic activity (i.e. autophosphorylation) and PKB activation.

Eto K et al. (1999) J Biol Chem "Ca(2+)/Calmodulin-dependent protein kinase cascade in Caenorhabditis elegans. Implication in transcriptional activation."

Kimura Y, Eto K, Tokumitsu H, Arai K, Takahashi N, Muramatsu M, Masuho Y

[J Biol Chem, 1999]

We have recently demonstrated that Caenorhabditis elegans Ca(2+)/calmodulin-dependent protein kinase kinase (CeCaM-KK) can activate mammalian CaM-kinase IV in vitro (Tokumitsu, H., Takahashi, N., Eto, K., Yano, S., Soderling, T.R., and Muramatsu, M. (1999) J. Biol. Chem. 274, 15803-15810). In the present study, we have identified and cloned a target CaM-kinase for CaM-KK in C. elegans, CeCaM-kinase I (CeCaM-KI), which has approximately 60% identity to mammalian CaM-KI. CeCaM-KI has 348 amino acid residues with an apparent molecular mass of 40 kDa, which is activated by CeCaM-KK through phosphorylation of Thr(179) in a Ca(2+)/CaM-dependent manner, resulting in a 30-fold decrease in the K(m) of CeCaM-KI for its peptide substrate. Unlike mammalian CaM-KI, CeCaM-KI is mainly localized in the nucleus of transfected cells because the NH(2)-terminal six residues ((2)PLFKRR(7)) contain a functional nuclear localization signal. We have also demonstrated that CeCaM-KK and CeCaM-KI reconstituted a signaling pathway that mediates Ca(2+)-dependent phosphorylation of cAMP response element-binding protein (CREB) and CRE-dependent transcriptional activation in transfected cells, consistent with nuclear localization of CeCaM-KI. These results suggest that the CaM-KK/CaM-KI cascade is conserved in C. elegans and is functionally operated both in vitro and in intact cells, and it may be involved in Ca(2+)-dependent nuclear events such as transcriptional activation through phosphorylation of CREB.

Awadzi K et al. (1986) Br J Clin Pharmacol "The effect of moderate urine alkalinisation on low dose diethylcarbamazine therapy in patients with onchocerciasis."

Orme ML, Edwards G, Breckenridge AM, Awadzi K, Adjepon-Yamoah KK, Gilles HM

[Br J Clin Pharmacol, 1986]

Twenty-one patients with moderate to heavy infections with O. volvulus were treated with 25 mg of diethylcarbamazine (DEC) citrate twice daily for 10 days. In 11 patients the urine was made alkaline with sodium bicarbonate, 2 g, administered 6 hourly for three doses daily beginning 1 day before DEC was started and continued throughout the DEC therapy. Ten patients served as controls. The mean pre-dose plasma DEC concentration during treatment and the mean plasma DEC half-life were significantly higher in bicarbonate treated patients as compared to controls. Total urinary excretion of DEC was significantly less in the bicarbonate treated group than in controls. Mean overall total reaction was higher in bicarbonate-treated patients but the difference was not significant. The bicarbonate-treated group achieved a significantly greater reduction in skin microfilarial counts than the control group as assessed 1 week after completion of therapy, but there was little difference at 1 month. Microfilarial killing was associated with microfilarial mobilisation, alteration in peripheral leucocytes and elevation in serum aminotransferases in both groups. There was no effect of DEC on the number of adult worms recovered in nodules removed at the end of the therapy. This study indicates that moderate urinary alkalinisation alters the kinetics of DEC and the therapeutic response. However the severity of clinical reaction coupled with the inadequate level of microfilarial killing achieved make it unlikely that manipulation of urinary pH will be of practical value in onchocerciasis chemotherapy.