一种新型丝氨酸苏氨酸激酶CPK的初步克隆及功能

从人胎肝cDNA文库中分离到一种cDNA ,推测的蛋白氨基酸序列具有明显的丝氨酸苏氨酸激酶结构域,可能编码一种新的丝氨酸苏氨酸激酶(命名为CPK ,cellproliferationassociationkinase) .CPKmRNA全长约3 0kb .RT PCR检测表明CPKmRNA在增殖活跃组织或细胞如人胚胎组织、肿瘤细胞株中呈高丰度表达,在成人组织则呈低丰度或不表达.利用PCR技术扩增大鼠同源cDNA ,以此为探针,Northern杂交发现CPK表达于多种成年小鼠组织,以脑组织丰度最高.小鼠2 3肝部分切除可迅速诱导CPKmRNA的表达,在术后2 4～4 8h达高峰,与2 3肝部分切除后细胞增殖期吻合.以小鼠同源cDNA片段为模板,合成RNA探针,原位杂交显示在胚胎发育期CPK低丰度表达在大多数组织,以神经系统组织变化最大,在第8d胚胎神经管内皮细胞中即出现表达,11～13d在端脑、脑膜、间脑、脊神经节表皮细胞、海马、小脑神经胶质细胞等多种神经细胞中表达且丰度较高,16d后这些组织的表达迅速下降到较低水平,表明CPK可能与神经系统的生长发育有一定关联.利用信号通路检测技术,观察到CPK的表达对MAPK ,p38MAPK途径的激活有明显的影响,并可显著增强表皮生长因子对这两条途径的激活,提示该激酶可能参与细胞因子的信号转导.     

Cloning and characterization of Hunk, a novel mammalian SNF1-related protein kinase

We previously identified a novel protein kinase, Hunk, by means of a degenerate PCR screen designed to isolate kinases expressed in the murine mammary gland. We now describe the molecular cloning, chromosomal localization, and activity of this kinase and characterize its spatial and temporal pattern of expression in the mouse. We have isolated a 5.0-kb full-length cDNA clone that contains the 714-amino-acid open reading frame encoding Hunk. Analysis of this cDNA reveals that Hunk is most closely related to the SNF1 family of serine/threonine kinases and contains a newly described SNF1 homology domain. Accordingly, antisera specific for Hunk detect an 80-kDa polypeptide with associated phosphotransferase activity. Hunk is located on distal mouse chromosome 16 in a region of conserved synteny with human chromosome 21q22. During fetal development and in the adult mouse, Hunk mRNA expression is developmentally regulated and tissue-specific. Moreover, in situ hybridization analysis reveals that Hunk expression is restricted to subsets of cells within a variety of organs in the adult mouse. These findings suggest a role for Hunk in murine development.     

Protein kinase C isoenzyme: selective expression pattern of protein kinase C-θ during mouse development

Protein kinase C (PKC)-θ, a serine/threonine protein kinase and novel PKC subfamily member, has been recently identified as an essential component of the T cell synapse which activates the NF-kB signaling cascade leading to expression of the IL-2 gene during T cell activation. By RNA in situ hybridization to whole-body embryo sections it is shown that the murine PKCθ is specifically expressed in tissues with hematopoietic and lymphopoietic activity. Expression is also evident in skeletal muscle. A further highly specific expression was observed in the peripheral and central nervous system which is described in detail. Expression in the brain persists up to adult stages.     

Water-deficit-responsive proteins in maritime pine

We have isolated three receptor-like kinase cDNAs from an Arabidopsis flower cDNA library by PCR using degenerate oligonucleotide primers for conserved domains of protein kinases. Cloning and sequencing of the full-length cDNAs, designated RKF1 to 3 (receptor-like kinase in flowers), showed that the putative extracellular domain of the RKF1 protein contains 13 tandem repeats of leucine-rich sequences and those of RKF2 and RKF3 have no significant homology with other plant sequences. RNA blot analysis revealed that the RKF1 mRNA is highly expressed in stamens while RKF2 and RKF3 mRNAs are present at low levels in all organs examined. In situ localization experiments indicated that the RKF1 mRNA is detectable in early flower primordia and during stamen development. In addition, when fused to a GUS reporter gene, the RKF1 promoter directed high GUS expression in pollen grains. Recombinant RKF1, produced in Escherichia coli, was found to have kinase activity with serine/threonine specificity in vitro.     

A mammalian dual specificity protein kinase, Nek1, is related to the NIMA cell cycle regulator and highly expressed in meiotic germ cells.

Screening of mouse cDNA expression libraries with antibodies to phosphotyrosine resulted in repeated isolation of cDNAs that encode a novel mammalian protein kinase of 774 amino acids, termed Nek1. Nek1 contains an N-terminal protein kinase domain which is most similar (42% identity) to the catalytic domain of NIMA, a protein kinase which controls initiation of mitosis in Aspergillus nidulans. In addition, both Nek1 and NIMA have a long, basic C-terminal extension, and are therefore similar in overall structure. Despite its identification with anti-phosphotyrosine antibodies, Nek1 contains sequence motifs characteristic of protein serine/threonine kinases. The Nek1 kinase domain, when expressed in bacteria, phosphorylated exogenous substrates primarily on serine/threonine, but also on tyrosine, indicating that Nek1 is a dual specificity kinase with the capacity to phosphorylate all three hydroxyamino acids. Like NIMA, Nek1 preferentially phosphorylated beta-casein in vitro. In situ RNA analysis of nek1 expression in mouse gonads revealed a high level of expression in both male and female germ cells, with a distribution consistent with a role in meiosis. These results suggest that Nek1 is a mammalian relative of the fungal NIMA cell cycle regulator.