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A protein activator of Ca2+/calmodulin (CaM)-dependent protein kinase I was purified from rat brain. The activator was retained on a CaM-Sepharose column in the presence of Ca2+ and kinase assay of renatured gel revealed the 64 kDa molecule in the purified activator fraction to be autophosphorylated and to phosphorylate recombinant CaM kinase I in the presence of Ca2+/calmodulin. These results suggest that this activator of CaM kinase I is also a CaM-dependent protein kinase. Phosphorylation of CaM kinase I by the activator resulted in drastic potentiation of its CaM-dependent activity. Furthermore, kinetic analyses demonstrated that the activation decreases the K_m values of CaM kinase I for both ATP and syntide-2 without a change in V_max values. Considering the quite low enzymatic activity of recombinant CaM kinase I without activation, the 64 kDa species might be essential for CaM kinase I function in vivo.Copyright 1995, 1999 Academic Press, Inc.

We earlier confirmed that there are isoforms of Ca2+/calmodulin (CaM)-dependent protein kinase I (CaM kinase I) (CaM kinase Ibeta1 and Igamma) beside CaM kinase Ialpha by cDNA cloning (Yokokura, H., Terada, O., Naito, Y., and Hidaka, H. (1997) Biochim. Biophys. Acta 1338, 8-12). Here, we demonstrate the existence of an isoform-specific activation mechanism of CaM kinase I and alternative splicing specifically regulating CaM kinase I (CaM kinase Ibeta2) in the central nervous system. To cast light on isoform structure-enzyme activity relationships, CaM kinase Ibeta1, Ibeta2, and Ialpha were expressed separately using a baculovirus/Sf9 cell expression system. The novel CaM kinase Ibeta2 isoform demonstrated similar catalytic activity to those of CaM kinase Ibeta1 and Ialpha. Interestingly, CaM kinase Ibeta1 and Ibeta2 both can activate CaM kinase Ialpha activity via phosphorylation at Thr177. Reverse transcribed-polymerase chain reaction analysis showed that CaM kinase Ibeta2 is dominant in the cerebrum and cerebellum, whereas CaM kinase Ibeta1 is present in peripheral tissues such as liver, heart, lung, kidney, spleen, and testis. CaM kinase Ibeta2 was also detected with an anti-CaM kinase Ibeta2 antibody in PC12 cells. The results indicate that alternative splicing is a means for tissue-specific expression of CaM kinase Ibeta. Thus the Thr177 residue of CaM kinase Ialpha is phosphorylated by not only CaM kinase kinase but also CaM kinase Ibeta for activation of the enzyme.

Autophosphorylation of Ca2+/calmodulin-dependent protein kinase V (CaM kinaseV) resulted in a drastic potentiation of the Ca2+/calmodulin-dependent activity, but Ca2+/calmodulin-independent activity was not generated. The rate of autophosphorylation increased with increases in the enzyme concentration, thereby suggesting intermolecular reactions. In the course of these investigations, another factor by which CaM kinase V is also phosphorylated and activated became evident. The addition of EGTA blocked CaM kinase V phosphorylation. The autophosphorylated CaM kinase V was not phosphorylated by the activator. These observations suggest that the activation factor is a CaM kinase which phosphorylates CaM kinase V at the autophosphorylation site, thereby potentiating the enzymatic activity.

Recently we obtained evidence for the existence of Ca2+/calmodulin-dependent protein kinase kinase(CaMKK), which was partially purified in the process of the purification step of Ca2+/calmodulin-dependent protein kinase V (CaM kinase V). This enzyme promoted phosphorylation of the autophosphorylation site on CaM kinase V and activated the activity(Mochizuki, H. et al. (1993) Biochem. Biophys. Res. Commun. 197, 1595-1600). Present study revealed that CaMKK also phosphorylated CaM kinase IV associated with the activation of its activity. Phosphorylation of CaM kinase IV by CaMKK occurred on multiple sites. Furthermore, CaM kinase IV and CaM kinase V phosphorylated each other which resulted in their activation. The phosphorylation site of CaM kinase V by CaM kinase IV was the same as the autophosphorylation site. Our study suggests the existence of a heretofore CaM kinases cascade consisting of a sequential activation of each CaM kinase. [formula]

Non-destructive elemental analysis with muonic X-rays was performed on human vertebral bone and lumbar torso phantoms. It can provide quantitative information on all elements in small deep-seated localized volumes. The experiment was carried out using the superconducting muon channel at TRIUMF in Vancouver, Canada and a lithium drifted germanium detector with an active area of 18.5 cm2. The muon channel produced backward-decayed negative muons with wide kinetic energy range from 0.5 to 54.2 MeV. The muon beam was collimated to a diameter of 18 mm. The number of incoming muons was about 4×l06∼5×l07per data point. In the measurements with human vertebral bones fixed with neutralized formaldehyde, the correlation coefficient between calcium content measured by muons and by atomic absorption analysis was 0.99 and the level of significance was 0.0003. In the measurements with lumbar torso phantoms, the correlation coefficient between calcium content measured by muons and by atomic absorption analysis was 0.99 and the level of significance was 0.02. The results suggest that elemental analysis in vertebral body trabecular bone using muonic X-rays closely correlates with measurements by atomic absorption analysis.

The MRI findings are described in a case of inflammatory pseudotumour of the bladder. The signal characteristics are correlated with the histological findings.