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Characterization of a Ca2+/calmodulin-dependent protein kinase cascade. Molecular cloning and expression of
H Tokumitsu1, H Enslen, T R Soderling
1Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.
Abstract:
Recent studies have demonstrated that Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) can mediate Ca(2+)-dependent regulation of gene expression through the phosphorylation of transcriptional activating proteins. We have previously identified and purified a 68-kDa rat brain CaM-kinase kinase that phosphorylates and increases total and Ca(2+)-independent activities of CaM-kinase IV (Tokumitsu, H., Brickey, D. A., Gold, J., Hidaka, H., Sikela, J., and Soderling, T. R. (1994) J. Biol. Chem. 269, 28640-28647). Using a partial amino acid sequence of the purified brain kinase, a CaM-kinase kinase cDNA was cloned from a rat brain cDNA library. Northern blot analysis showed that CaM-kinase kinase mRNA (3.4 kilobases) was expressed in rat brain, thymus, and spleen. Sequence analyses revealed that the cDNA encoded a 505-amino acid protein, which contained consensus protein kinase motifs and was 30-40% homologous with members of the CaM-kinase family. Expression of the cDNA in COS-7 cells yielded an apparent 68-kDa CaM-binding protein, which catalyzed in vitro activation in the presence of Mg2+/ATP and Ca2+/ CaM of CaM-kinases I and IV but not of CaM-kinase II. Co-expression of CaM-kinase kinase with CaM-kinase IV gave a 14-fold enhancement of cAMP-response element-binding protein-dependent gene expression compared with CaM-kinase IV alone. These results are consistent with the hypothesis that CaM-kinases I and IV are regulated through a unique signal transduction cascade involving CaM-kinase kinase.
Insights
Researchers identified and cloned a CaM-kinase kinase cDNA, revealing its role in regulating CaM-kinase IV activity. This kinase enhances gene expression, suggesting a unique signal transduction pathway for CaM-kinases I and IV.
Area of Science:
- Molecular Biology
- Neuroscience
- Signal Transduction
Background:
- Ca2+/calmodulin-dependent protein kinase IV (CaM-kinase IV) regulates gene expression via phosphorylation of transcription factors.
- A 68-kDa rat brain CaM-kinase kinase was previously identified, which activates CaM-kinase IV.
Purpose of the Study:
- To clone the CaM-kinase kinase cDNA.
- To characterize the function and expression of CaM-kinase kinase.
- To elucidate its role in the CaM-kinase signaling pathway.
Main Methods:
- Cloning of CaM-kinase kinase cDNA from a rat brain library using partial amino acid sequence.
- Northern blot analysis to determine mRNA expression patterns.
- Expression of cDNA in COS-7 cells and in vitro kinase assays.
- Co-expression studies with CaM-kinase IV to assess effects on gene expression.
Main Results:
- CaM-kinase kinase mRNA is expressed in rat brain, thymus, and spleen.
- The cloned cDNA encodes a 505-amino acid protein with kinase motifs, homologous to CaM-kinase family members.
- Expressed CaM-kinase kinase activates CaM-kinases I and IV, but not CaM-kinase II, in vitro.
- Co-expression with CaM-kinase IV significantly enhanced cAMP-response element-binding protein-dependent gene expression.
Conclusions:
- CaM-kinase kinase is a key component in the regulation of CaM-kinases I and IV.
- These findings support a novel signal transduction cascade involving CaM-kinase kinase for CaM-kinases I and IV regulation.