Baier G, Telford D, Giampa L, Coggeshall K M, Baier-Bitterlich G, Isakov N, Altman A
La Jolla Institute for Allergy and Immunology, California 92037.
J Biol Chem. 1993 Mar 5;268(7):4997-5004.
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.
丝氨酸/苏氨酸激酶的蛋白激酶C（PKC）家族成员在调节多种细胞类型的分化和生长中起关键作用，迄今为止，已报道克隆了七个编码八种不同亚型的哺乳动物PKC基因。在此，我们描述了一种新型PKC亚型（称为PKCθ）的cDNA的分子克隆和推导的一级结构，该cDNA是在试图鉴定在造血细胞中选择性表达的PKC基因的过程中分离得到的。在聚合酶链反应（PCR）中使用与保守序列基序相对应的简并寡核苷酸引物，这些基序将PKC家族与其他蛋白激酶区分开来，以从人外周血淋巴细胞衍生的cDNA文库中扩增推定的PKC基因的部分核心序列。对选定克隆的DNA测序揭示了几个与PKC相关的序列，其中一个基于与已知PKC亚型的序列比较，代表一种新型PKC亚型。通过锚定PCR克隆并对整个编码序列进行测序，使用来自人白血病T细胞系（Jurkat）的cDNA确定了完整的cDNA序列。在这个约2.7千碱基对的cDNA中包含一个2118个核苷酸的开放阅读框，编码一个推定的82 kDa蛋白。推导的一级结构包含蛋白激酶催化域的共有序列，基于其氨基酸序列和结构域结构，它是PKC家族的成员之一。PKCθ与PKCδ显示出最高的同源性，缺乏Ca(2+)结合C2结构域，因此属于不依赖Ca(2+)的PKC酶亚家族，该亚家族还包括δ、ε、ζ和η亚型。核糖核酸酶保护分析和半定量PCR分析表明，虽然PKCθ转录本在各处均有表达，但在造血组织和细胞系中水平最高，包括T细胞和胸腺细胞。相比之下，在脑和睾丸中的表达水平相当低，并且在几种人癌细胞系中未检测到转录本。针对独特的（V3结构域）细菌表达的PKCθ片段产生的兔抗血清特异性地从Jurkat细胞裂解物中免疫沉淀出一种82 kDa蛋白。因此，PKCθ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起作用。
