No abstract

A cDNA encoding the homolog of the human pre-Bcell colony-enhancing factor (PBEF), a cytokine-like secreted protein, was isolated from a rat cDNA library. This protein existed in both the cytoplasm and nucleus of the cells, and the amount was higher in the cytoplasm than in the nucleus of proliferating PC-12 and Swiss 3T3 cells but higher in the nucleus than in the cytoplasm of the PC-12 cells treated with nerve growth factor and the 3T3 cells grown to a con£uent state. Thus, the so-called PBEF is not a cytokine-like secreted protein but an intracellular protein associated with the cell cycle.

No abstract

Calmodulin-dependent protein kinase IV (CaM-kinase IV), which plays crucial roles in the functioning of Ca2+ in the central nervous and immune systems, is markedly activated upon phosphorylation through the action of CaM-kinase kinase. Our previous immunotitration analysis suggested the existence of an isoform different from CaM-kinase kinase alpha, the beta isoform, in rat brain [Okuno, S., Kitani, T., and Fujisawa, H. (1996) J. Biochem. 119, 1176-1181]. In the present study, cDNA for CaM-kinase kinase beta was cloned from a rat cerebellar cDNA library. The coded protein consisted of 587 amino acids with a molecular weight of 64,445. Western blot analysis revealed that CaM-kinase kinase beta significantly existed only in the brain. The enzyme was not significantly detected in the retina where CaM-kinase kinase alpha exists.

Activating transcription factor 1 (ATF1) and the cAMP response element-binding protein (CREB) are members of the CREB/ATF family implicated in cAMP- and calcium-induced transcriptional activation. Although ATF1 and CREB share extensive homology, the function of ATF1 is poorly understood. Its phosphorylation state and activation by Ca2+- and calmodulin-dependent protein kinase (CaMK) II were therefore examined. Phosphopeptide mapping analysis and Western blotting studies demonstrated that in vitro, CaMK II phosphorylates only Ser63 (corresponding to Ser133 of CREB), which is essential for the activation, and not Ser72 (corresponding to Ser142 of CREB), which is a negative regulation site. Both ATF1 and CREB bound CBP in a phosphorylation-dependent manner. As expected from these in vitro studies, transient transfection studies revealed that ATF1 is activated by CaMK II. Our findings suggest that CaMK II mediates transactivation of cAMP responsive genes via ATF1.

Calmodulin-dependent protein kinase phosphatase (CaMKP) dephosphorylates and concomitantly deactivates multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs), such as CaMKI, CaMKII, and CaMKIV. In the present study, a nuclear CaMKP-related protein, CaMKP-N, was identified. This protein consisted of 757 amino acid residues with a calculated molecular weight of 84,176. Recombinant CaMKP-N dephosphorylated CaMKIV. The activity of CaMKP-N requires Mn(2+) ions and is stimulated by polycations. Transiently expressed CaMKP-N in COS-7 cells was localized in the nucleus. This finding together with previous reports regarding localization of CaMKs indicates that CaMKP-N dephosphorylates CaMKIV and nuclear CaMKII, whereas CaMKP dephosphorylates CaMKI and cytosolic CaMKII.

The importance of the individual amino acid residues of AIP (KKALRRQEAVDAL), a highly specific inhibitor of calmodulin-dependent protein kinase II (CaMKII), was studied. Replacement of Arg T , Gln U , or Ala W by other amino acid residues produced a marked increase in the IC SH value. Leu R and Val IH were also sensitive to replacement, but some hydrophobic amino acids could substitute for these residues. Although replacement of Ala Q , Glu V , Ala IP , and Leu IQ by other residues produced no significant increase in the IC SH , the substitution of Lys for Ala Q decreased the IC SH . An AIP analog (KKKLRRQEAFDAY), in which Ala Q and Val IH were replaced with Lys and Phe, respectively, showed an IC SH value as low as 4 nM, suggesting that it is a useful tool for studying the physiological roles of CaMKII.z 1998 Federation of European Biochemical Societies.

Ca 2+ /Calmodulin-dependent protein kinase (CaM kinase) phosphatase, occurring in the cytoplasm of all tissues, dephosphorylates and thereby deactivates multifunctional CaM kinases, such as CaM kinases I, II and IV. In contrast, CaM kinase phosphatase N has been reported to occur almost exclusively in the brain and to be localized in the nucleus in the transfected COS-7 cells, as examined immunocytochemically with antibodies against the carboxyl-terminal segment of the enzyme, indicating its involvement in the deactivation of CaM kinase IV. Here, we show that the majority of the naturally occurring CaM kinase phosphatase N in the brain exists not in the intact form of the enzyme (83.4 kDa) but in a form (61.1 kDa) in which the carboxyl-terminal segment containing nuclear localization signals is deleted, and that it is present mostly in the cytoplasm but a little in the nucleus throughout the central nervous system, although occurring mostly in the nucleus in some large neurons. Strong immunostaining of the enzyme was also observed at postsynaptic density. These findings suggest that CaM kinase phosphatase N is involved in the regulation of not only CaM kinase IV but also CaM kinases II and I. Keywords: Ca Multifunctional Ca 2+ /calmodulin-dependent protein kinases (CaM kinases) such as CaM kinases I, II and IV play important roles in controlling a variety of cellular functions in response to an increase in intracellular Ca 2+ (Fujisawa 2001). Among the three CaM kinases, CaM kinase II is activated through autophosphorylation of Thr286 (Kwiatkowski et al. 1988;Ikeda et al. 1991;Katoh and Fujisawa 1991;Ishida et al. 1996) and the other two, CaM kinase IV (Okuno and Fujisawa 1993;Okuno et al. 1994;Tokumitsu et al. 1994) and CaM kinase I, (Mochizuki et al. 1993;Lee and Edelman 1994) are activated through phosphorylation of Thr196 and Thr177, respectively, by upstream CaM kinase kinases. The phosphorylation sites of the three CaM kinases are specifically dephosphorylated by CaM kinase phosphatase and consequently, the CaM kinases are deactivated (Ishida et al. 1998a,b;Kitani et al. 1999). An exclusive cytosolic localization of CaM kinase phosphatase in PC-12 cells (Kitani et al. 1999) and in the rat brain (Nakamura et al. 2000) has been demonstrated. On the other hand, nuclear CaM kinase phosphatase, CaM kinase phosphatase N, which shows catalytic properties similar to those of cytosolic CaM kinase phosphatase, has recently been identified (Takeuchi et al. 2001). In contrast to the widespread occurrence of CaM