Region Specific Expression Of CaMKⅡα Gene In Monkey Brain And Its Promoter Analysis

Multifunctional Calcium/calmodulin-dependent protein kinase II(CaMKII) participates in diverse calcium signaling pathways in neurons. The a subunit of CaMKII (CaMKIIα) is neuron-specific and highly expressed in the rat brain. To characterize specific expression pattern of CaMKIIα in monkey brain, we cloned and sequenced the CaMKIIα cDNA. Five distinct isoforms of CaMKIIα were isolated. Two of them were characterized as CaMKIIα and CaMKIIα_B. The other three new isoforms were named α3, α3_B and α4. Among them, α3 and α3_B have a 171-bp deletion relative to the a and α_B isoforms, respectively, and α4 has a 76-bp deletion relative to the a isoform. These alternations are located within the variable region. Using RT-PCR with specific primers flanking the variable region, the expression of α3 and α3_B were confirmed in monkey brain, but not in rat or dog brain.The distribution of CaMKIIα in frontal cortex, hippocampus, thalamus, midbrain, and cerebellum in monkey brain were examined by RT-PCR and western blot. The highest mRNA expression level in frontal cortex and the highest protein expression level in thalamus revealed regional differences in gene expression and the different protein stability of a and α_B isoforms.The promoter of the rat CaMKIIα gene was identified to contain an essential TATA element. Cell-based functional assay showed that the rat promoter displayed greater activity in neuronal cells than in non-neuronal cells. To characterize the human CaMKIIα promoter, we developed a promoter-reporter gene assay using different cell lines. A 2047-bp human CaMKIIα promoter was cloned from human genomic DNA. Unlike the rat CaMKIIα promoter, DNA sequence analysis showed that the human promoter was devoid of TATA element. We made series deletions of the promoter and fused the mutant promoters to a luciferase reporter gene. The promoter-reporter constructs were transfected into human neuroblastoma SH-SY5Y and pheochromocytoma PC12 neuronal cell lines as well as human embryonic kidney HEK293 and human glioma U251 non-neuronal cell lines. The reporter gene assay...