Baier G, Baier-Bitterlich G, Meller N, Coggeshall K M, Giampa L, Telford D, Isakov N, Altman A
La Jolla Institute for Allergy and Immunology.
Eur J Biochem. 1994 Oct 1;225(1):195-203. doi: 10.1111/j.1432-1033.1994.00195.x.
In this study, the recently identified human protein kinase C-theta (PKC-theta) isoform has been biochemically characterized in detail. An antiserum raised against the unique V3 domain of PKC-theta identified an 80-kDa protein in all human T-cell lines tested, in erythroleukemia K562 cells and in histiocytic lymphoma U-937 cells, but not in a B-lymphoma line (Raji) or in several melanoma, carcinoma, schwanoma or astrocytoma lines, confirming, at the protein level, its predominant expression in hematopoietic cell lines, in particular T cells. Immunoreactive PKC-theta was detected almost exclusively in the cytosolic compartment of unstimulated Jurkat T cells. Stimulation with phorbol ester, however, caused rapid translocation to the membrane. In order to compare the properties of PKC-theta with a representative member of the Ca(2+)-dependent PKC enzymes, full-length cDNAs encoding PKC-theta or PKC-alpha were transiently expressed in COS-1 cells, and recombinant enzymes were partially purified via a six-histidine peptide tag. The catalytic activity of these PKC enzymes was assayed against distinct substrates in the absence and presence of known PKC cofactors. Significant differences were found with respect to activation requirements and substrate preferences between PKC-theta and PKC-alpha. Both enzymes were stimulated by phospholipid and phorbol ester, and were active towards a PKC-derived substrate peptide corresponding to the pseudosubstrate site of PKC. In contrast to PKC-alpha, however, full activation of PKC-theta did not require Ca2+, and its basal activity towards histone H1 was not stimulated by lipid cofactors. Additionally, a myelin-basic-protein-(MBP)-derived peptide, which was readily phosphorylated by PKC-alpha, was a poor substrate for PKC-theta. Similar to PKC-alpha, transient PKC-theta overexpression in murine EL4 thymoma cells caused an approximately 2.5-fold increase in the phorbol-12-myristate-13-acetate-induced transcriptional activation of an interleukin-2 promoter-reporter gene construct. The unique expression and functional properties of PKC-theta suggest that it may play a specialized role in T-cell signaling pathways.
在本研究中，最近鉴定出的人蛋白激酶C-θ（PKC-θ）同工型已得到详细的生化特性分析。针对PKC-θ独特的V3结构域产生的抗血清，在所有测试的人T细胞系、红白血病K562细胞和组织细胞淋巴瘤U-937细胞中鉴定出一种80 kDa的蛋白质，但在B淋巴瘤细胞系（Raji）以及几种黑色素瘤、癌、神经鞘瘤或星形细胞瘤细胞系中未检测到，这在蛋白质水平上证实了其在造血细胞系，特别是T细胞中的主要表达。免疫反应性PKC-θ几乎仅在未刺激的Jurkat T细胞的胞质区室中检测到。然而，佛波酯刺激导致其迅速转位至细胞膜。为了将PKC-θ的特性与钙依赖性PKC酶的代表性成员进行比较，编码PKC-θ或PKC-α的全长cDNA在COS-1细胞中瞬时表达，重组酶通过六组氨酸肽标签进行部分纯化。在有无已知PKC辅因子的情况下，针对不同底物测定了这些PKC酶的催化活性。发现PKC-θ和PKC-α在激活要求和底物偏好方面存在显著差异。两种酶均受到磷脂和佛波酯的刺激，并且对对应于PKC假底物位点的PKC衍生底物肽具有活性。然而，与PKC-α不同，PKC-θ的完全激活不需要Ca2+，并且其对组蛋白H1的基础活性不受脂质辅因子的刺激。此外，一种容易被PKC-α磷酸化的髓鞘碱性蛋白（MBP）衍生肽，是PKC-θ的不良底物。与PKC-α类似，在小鼠EL4胸腺瘤细胞中瞬时过表达PKC-θ导致佛波醇-12-肉豆蔻酸酯-13-乙酸酯诱导的白细胞介素-2启动子-报告基因构建体的转录激活增加约2.5倍。PKC-θ独特的表达和功能特性表明它可能在T细胞信号通路中发挥特殊作用。
