| Calcium, an intracellular second messenger, is known to be required for cells growth and differentiation. A number of studies have shown that this requirement for calcium during cell division is mediated by calmodulin (CaM), an intracellular calcium-binding protein. Ca 2+/calmodulin-dependent protein kinase II (CaMKII), a target of calcium/CaM, is a multifunctional calcium/calmodulin-dependent serine/threonine protein kinase II that has broad substrate specificity and wide distribution in mammalian tissue. In vitro it can phosphorylate up to 40 proteins, including enzymes, ion channels, transcription factors, etc. and a number of these proteins appear to be physiological substrates.Recent studies suggest that CaMKII plays an important role in cell growth, proliferation and differentiation. CaMKII inhibitor is an useful tool in the study of CaMKII physiological function. CaMKII inhibitor can inhibit CaMKII activity by connecting with Ca2~VCaM binding site or affecting its catalysis. This inhibition of enzyme activity can block the phospharylation of substrate by CAMKII or regulate certain gene transcription, resulting in the induction of tumor cell apoptosis or necrosis. In the present study, a novel CaMKII inhibitor, designated as hCaMKIINa, was identified from human fetal brain.Part I Cloning, sequence analysis and mRNA expression pattern analysis of hCaMKIINaIn 2001, the first human endogenetic CaMKII inhibitory protein, designated as human CaMKII inhibitor beta (hCaMKIINp), was identified in our lab by large-scale sequencing of human dendritic cell cDNA library. Another novel sequence was identified by homology search using hCaMKIINp cDNA sequence and in silico assembly ofexpression sequence tags (ESTs), and finally obtained by PCR amplification from human fetal brain. The full-length cDNA was 860bp, encoding a protein of 78 residues with a molecular mass of 8556.62 Dalton and an isoelectric point of 5.22. Homology analysis showed that the novel protein was greatly similar to CaMKII inhibitory protein family members, especially rat CaMKIINa. According to the nomenclature of CaMKII inhibitory protein family and the significant homology with rat CaMKIINa, the novel molecule was named human CaMKII inhibitory protein alpha (hCaMKIINa). The hCaMKIINa gene was mapped in Chromosome Ip36.13. The sequence of hCaMKIINa has been submitted to GenBank database (accession No. AY204901) and applied for National Invention Patent (application No. 03151428.6). Sequence analysis revealed the presence of a conserved 27-residue inhibitory region in the C-terminal of hCaMKIINa protein. Previous studies have suggested that the 27-residue conserved inhibitory region may play key roles in the inhibition of CaMKII activity by CaMKIIN.By RT-PCR, we have demonstrated the mRNA expression of hCaMKIINa in some solid tumor cells, including breast adenocarcinoma MCF-7, glioblasoma A172, cerix epitheloid carcinoma HeLa, hepatoma SMMC7721, prostate adenocarcinoma PC-3, lung carcinoma A549 and ovary adenocarcinoma CaoV-3 cells. However, the Colon adenocarcinoma cell lines LoVo and HT-29 do not express hCaMKIINa. hCaMKIINa mRNA was also detected in hematopoietic tumor cells, including histiocytic lymphoma U937, chronic myelogenous leukemia K562, Burkitt's lymphoma Daudi cells. The mRNA expression pattern of hCaMKIINa in normal human adult tissues was examined by PCR. The result showed that hCaMKIINa had wide distribution in normal human tissues, including heart, brain, spleen, kidney, placenta, lung, liver, ovary, testis, small intestine, colon, prostate, thymus, pancreas, but no expression was detected in skeletal muscle.Part II Interaction of hCaMKIINa and CaMKII and functional analysis of the 27-residue conserved inhibitory regionSequence and homology analysis has suggested that hCaMKIINa be a member ofCaMKII inhibitor family. Then we wonder whether hCaMKIINa could act as a functional inhibitor of CaMKII through specifically interacting with CaMKII and inhibiting CaMKII activity. To determine the interaction of hCaMKIINa w...
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