Molecular cloning and characterization of PKC theta, a novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells.
Molecular cloning and characterization of PKC theta, a novel member of the protein kinase C (PKC) gene family expressed predominantly in hematopoietic cells.
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DOI：
10.1016/s0021-9258(18)53494-3
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发表时间：
1993-03
期刊：
影响因子：
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通讯作者：
G. Baier;D. Telford;L. Giampa;K. Coggeshall;G. Baier‐Bitterlich;N. Isakov;A. Altman
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文献类型：
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作者：
G. Baier;D. Telford;L. Giampa;K. Coggeshall;G. Baier‐Bitterlich;N. Isakov;A. Altman
丝氨酸/苏氨酸激酶的蛋白激酶 C (PKC) 家族成员在调节不同细胞类型的分化和生长中发挥着关键作用，迄今为止，已报道了编码八种不同亚型的七个哺乳动物 PKC 基因的克隆。在这里，我们描述了编码新型 PKC 同种型（称为 PKC theta）的 cDNA 的分子克隆和推导的一级结构，该同种型是在尝试鉴定在造血细胞中选择性表达的 PKC 基因的过程中分离出来的。与保守序列基序相对应的简并寡核苷酸引物将 PKC 家族与其他蛋白激酶区分开来，用于聚合酶链式反应 (PCR)，从人外周血淋巴细胞衍生的 cDNA 文库中扩增推定 PKC 基因的部分核心序列。对选定克隆的 DNA 测序揭示了几个 PKC 相关序列，其中一个序列基于与已知 PKC 同工型的序列比较，代表了一种新的 PKC 同工型。使用源自人白血病 T 细胞系 (Jurkat) 的 cDNA，通过锚定 PCR 克隆和测序整个编码序列来确定完整的 cDNA 序列。这个大约 2.7 kDa 的 cDNA 中包含一个由 2,118 个核苷酸组成的开放阅读框，编码假定的 82 kDa 蛋白质。推导的一级结构包含蛋白激酶催化结构域特征的共有序列，并且基于其氨基酸序列和结构域结构，它是 PKC 家族的成员。 PKC theta 与 PKC delta 表现出最高的同源性，缺乏 Ca(2+) 结合 C2 结构域，因此属于 Ca(2+) 独立 PKC 酶亚家族，该酶还包括 delta、epsilon、zeta 和 eta 亚型。 RNase 保护测定和半定量 PCR 分析表明，尽管 PKC theta 转录本普遍表达，但在造血组织和细胞系（包括 T 细胞和胸腺细胞）中表达水平最高。相比之下，大脑和睾丸中的表达水平要低得多，并且在几种人类癌细胞系中没有检测到转录本。针对独特的（V3 结构域）细菌表达的 PKC theta 片段产生的兔抗血清可特异性免疫沉淀 Jurkat 细胞裂解物中的 82-kDa 蛋白。因此，PKC theta 代表 PKC 家族的另一个成员，其在造血细胞中的主要表达表明它可能在这些细胞特有的信号转导和生长调节途径中发挥作用。
Members of the protein kinase C (PKC) family of serine/threonine kinases play a key role in regulating the differentiation and growth of diverse cell types and, to date, the cloning of seven mammalian PKC genes encoding eight distinct isoforms has been reported. Here we describe the molecular cloning and deduced primary structure of a cDNA encoding a novel PKC isoform, termed PKC theta, which was isolated in the course of attempts to identify PKC genes that are expressed selectively in hematopoietic cells. Degenerate oligonucleotide primers corresponding to conserved sequence motifs, which distinguish the PKC family from other protein kinases, were employed in polymerase chain reactions (PCR) to amplify partial core sequences of putative PKC genes from a human peripheral blood lymphocyte-derived cDNA library. DNA sequencing of selected clones revealed several PKC-related sequences, including one that, on the basis of sequence comparison with known PKC isoforms, represented a novel PKC isoform. The complete cDNA sequence was determined by anchored PCR cloning and sequencing the entire coding sequence, using cDNA derived from a human leukemic T cell line (Jurkat). Included within this approximately 2.7-kilobase pair cDNA is an open reading frame of 2,118 nucleotides encoding a putative 82-kDa protein. The deduced primary structure contains consensus sequences characteristic of protein kinase catalytic domains and, based on its amino acid sequence and domain structure, is a member of the PKC family. PKC theta displays the highest homology to PKC delta, lacks the Ca(2+)-binding C2 domain and, thus, belongs to the subfamily of Ca(2+)-independent PKC enzymes which also includes the delta, epsilon, zeta, and eta isoforms. RNase protection assays and semiquantitative PCR analysis indicated that, although PKC theta transcripts are expressed ubiquitously, the highest levels are found in hematopoietic tissues and cell lines, including T cells and thymocytes. In contrast, the expression levels in the brain and testes are considerably lower, and no transcripts were detected in several human carcinoma cell lines. A rabbit antiserum raised against a unique (V3 domain) bacterially expressed PKC theta fragment immunoprecipitated specifically an 82-kDa protein from Jurkat cell lysates. Thus, PKC theta represents an additional member of the PKC family, and its predominant expression in hematopoietic cells suggests that it may play a role in signal transduction and growth regulatory pathways unique to these cells.