斑馬魚中Camodulin-dependent protein kinase II inhibitor蛋白的選殖、表現模式及功能分析

Abstract

Ca2+/Calmodulin-dependent protein kinase II (CaMKII) plays a pivotal role in a wide range of cellular function including cell cycle and neuron development, and signal transduction. Recently, two proteins, termed Ca2+/Calmodulin-depent protein kinase inhibitor (CaMKIIN) α and β, specific bound to and inhibited CaMKII activity has been reported in vertebrate. The role of CaMKIINs in development still remained to be explored. Herein, we demonstrated the isolation and expression patterns of zebrafish CaMKIINs, named as camkiin 1 and camkiin 2. camkiin 1 and camkiin 2 gene both encoded 79 amino acids and shared over 70% similarity to corresponded genes in mammalian, amphibian and others fish species. Multiple protein alignment indicated that the 27 residues conserved inhibitory region (CIR) shared highest homology. Semi-quantitative RT-PCR analysis revealed that both genes were weakly or undetectable before five somites stages whereas enriched in 24 hour post-fertilization (hpf) and persistently expressed thereafter. Both camkiin 1 and camkiin 2 were found to be highly expressed in adult eyes and brain. RNA transcripts of camkiin 1 were prominently expressed in forebrain and hindbrain region, especially in telencephalon. camkiin 2 was detected in anterior brain region and hindbrain nucleous. Through GST-pull down, co-immunoprecipitation and kinase assay, we have demonstrated that zcamkiins can bind to and reduce CaMKII activity. Morpholino antisense oligonucleotides were used to knockdown the zcamkiin expression. Both camkiin morphant have midbrain hidbrain boundary (MHB) abnormity. camkiin 1 morphant resulted in midbrain defect and enlarged hindbrain. Small eyes and flat head were found in camkiin 2 morphant. No synergistical effects were observed in morphont knockdown of both genes. Malformation of MHB and reduction of neurogenin-1 signals were observed in camkiin 1 but not camkiin 2 morphants. Co-injected with camkiin 1 mRNA can rescue the deformities. However, no significant alternation of markers for forebrain, midbrain, and hidbrain was found in camkiin 2 morphants. Taken together, our results provided the first evidences of identification and expression of zebrafish camkiin 1 and camkiin 2 genes. Distinct spatial expression pattern of camkiin 1 and camkiin 2 were observed by whole-mount in situ hybridization. Our results also suggested that these two genes may play an important role in zebrafish brain development.