Molecular cloning and characterization of the human protein kinase D2 -: A novel member of the protein kinase D family of serine threonine kinases
Molecular cloning and characterization of the human protein kinase D2 -: A novel member of the protein kinase D family of serine threonine kinases
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DOI：
10.1074/jbc.m008719200
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发表时间：
2001-02-02
影响因子：
4.8
通讯作者：
Seufferlein, T
中科院分区：
文献类型：
--
作者：
Sturany, S;Van Lint, J;Seufferlein, T
我们分离到了一个新的人丝氨酸苏氨酸蛋白激酶基因的全长cDNA。推导出的蛋白质序列在N端包含两个富含半胱氨酸的基序，一个pleckstrin同源结构域和一个包含丝氨酸蛋白激酶所有特征序列基序的催化结构域。它与丝氨酸苏氨酸蛋白激酶PKD/PKC mu和PKC nu的同源性最强，特别是在富含半胱氨酸的双锌指状基元、pleckstrin同源结构域和蛋白激酶结构域，而与PKC家族的其他成员只有低程度的序列相似性，因此，新蛋白被命名为蛋白激酶D2 （PKD2）。PKD2的mRNA在人和小鼠组织中广泛表达。它在sds -聚丙烯酰胺凝胶电泳中编码一个分子量为105 kDa的蛋白，该蛋白在包括HL60细胞在内的多种不表达PKC mu的人类细胞系中表达。活体磷酯结合研究表明[H-3]磷酯12,13-二丁酸酯与PKD2的结合具有浓度依赖性。在二油基磷脂酰丝氨酸存在的情况下，添加1,13 -二丁酸磷以协同方式刺激PKD2的自磷酸化。佛波酯也能刺激完整细胞中PKD2的自磷酸化。磷酸酯激活的PKD2可有效磷酸化外源底物组蛋白H1。此外，我们可以确定c端Ser（876）残基是PKD2体内磷酸化位点。PKD2的Ser（876）磷酸化与激酶的激活状态相关。最后，胃泌素被发现是稳定转染CCKB/胃泌素受体的人AGS-B细胞中PKD2的生理激活剂。因此，PKD2是一种新的磷酯和生长因子刺激的蛋白激酶。
We have isolated the full-length cDNA of a novel human serine threonine protein kinase gene. The deduced protein sequence contains two cysteine-rich motifs at the N terminus, a pleckstrin homology domain, and a catalytic domain containing all the characteristic sequence motifs of serine protein kinases. It exhibits the strongest homology to the serine threonine protein kinases PKD/PKC mu, and PKC nu, particularly in the duplex zinc finger-like cysteine-rich motif, in the pleckstrin homology domain and in the protein kinase domain, In contrast, it shows only a low degree of sequence similarity to other members of the PKC family, Therefore, the new protein has been termed protein kinase D2 (PKD2). The mRNA of PKD2 is widely expressed in human and murine tissues. It encodes a protein with a molecular mass of 105 kDa in SDS-polyacrylamide gel electrophoresis, which is expressed in various human cell lines, including HL60 cells, which do not express PKC mu. lit vivo phorbol ester binding studies demonstrated a concentration-dependent binding of [H-3]phorbol 12,13-dibutyrate to PKD2. The addition of phorbol 12,13-dibutyrate in the presence of dioleoylphosphatidylserine stimulated the autophosphorylation of PKD2 in a synergistic fashion. Phorbol esters also stimulated autophosphorylation of PKD2 in intact cells. PKD2 activated by phorbol esters efficiently phosphorylated the exogenous substrate histone H1. In addition, we could identify the C-terminal Ser(876) residue as an in vivo phosphorylation site within PKD2. Phosphorylation of Ser(876) of PKD2 correlated with the activation status of the kinase. Finally, gastrin was found to be a physiological activator of PKD2 in human AGS-B cells stably transfected with the CCKB/gastrin receptor. Thus, PKD2 is a novel phorbol ester- and growth factor-stimulated protein kinase.