Identification of the isoforms of Ca(2+)/Calmodulin-dependent protein kinase II in rat astrocytes and their subcellular localization.
Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) occurs in astrocytes as well as in neurons in brain. We have reported that CaM kinase II is involved in the regulation of cytoskeletal proteins and gene expression in astrocytes. In this study, we identified all isoforms of CaM kinase II in astrocytes and examined their subcellular localization. When we amplified the isoforms of four subunits by RT-PCR followed by the "nested" PCR, totally 10 isoforms were obtained. Immunoblot analyses with five types of antibodies against CaM kinase II indicated that the most abundant isoform was delta2. Immunostaining suggested that the delta2 isoform was localized predominantly at the Golgi apparatus. The localization of the delta2 isoform at the Golgi apparatus was also observed in NG108-15 cells. We overexpressed all isoforms that contained the nuclear localization signal to examine their nuclear targeting in NG108-15 cells. In contrast to the alphaB and delta3 isoforms that entered the nucleus, as reported, the gammaA isoform was excluded from the nucleus in the transfected NG108-15 cells. These results suggest that the 15-amino acid insertion following the nuclear localization signal inhibits the nuclear targeting of the gammaA isoform.
Takeuchi
Y
Y
Department of Pharmacology, Kumamoto University School of Medicine, Kumamoto, Japan.
Yamamoto
H
H
Fukunaga
K
K
Miyakawa
T
T
Miyamoto
E
E
D000595:Amino Acid Sequence; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology*; D015152:Blotting, Northern; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D002417:Cattle; D002467:Cell Nucleus / Q000201:enzymology / Q000235:genetics; D017931:DNA Primers; D015971:Gene Expression Regulation, Enzymologic; D005910:Glioma; D006056:Golgi Apparatus / Q000201:enzymology; D007527:Isoenzymes / Q000032:analysis / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis / Q000502:physiology; D019913:Nuclear Localization Signals / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D014407:Tumor Cells, Cultured
D017931:DNA Primers; D007527:Isoenzymes; D019913:Nuclear Localization Signals; D012333:RNA, Messenger; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases