Chow L M, Davidson D, Fournel M, Gosselin P, Lemieux S, Lyu M S, Kozak C A, Matis L A, Veillette A
McGill Cancer Centre, McGill University, Montréal, Canada.
Oncogene. 1994 Dec;9(12):3437-48.
Recently, we and others have cloned cDNAs encoding a second member of the Csk family of inhibitory tyrosine protein kinases, which we have termed Ntk. Intriguingly, the mouse ntk cDNA sequences published by two independent groups differed by the presence or absence of a 136 nucleotide-insert near their 5' ends. In this report, we demonstrate that this 136 nucleotide-sequence likely corresponds to a complete exon in the ntk gene (termed exon 2), and that the two types of cDNAs/transcripts are produced by alternative splicing. Using ribonuclease protection assays, it was also established that brain and lymphoid organs, as well as most hemopoietic cells, predominantly expressed ntk transcripts lacking exon 2. In contrast, selected hemopoietic cell lines, such as the immature myeloid cell lines 32D cl3(G) and WEHI-3B, exclusively possessed exon 2-bearing RNAs. Interestingly, exon 2 introduced a novel in-frame upstream AUG in the ntk transcript, which is in the appropriate context for translation initiation. Evidence was obtained that this AUG is utilized in vivo, and that it extends the amino-terminal sequence of Ntk by 40 amino acids. Indeed, while exon 2-deficient ntk RNAs were translated into a 52 kilodalton (kDa) polypeptide (p52ntk), those bearing exon 2 produced a 56 kDa protein (p56ntk). Furthermore, p56ntk, but not p52ntk, was recognized by an antiserum directed against the novel amino-terminal sequence encoded by exon 2. Additional biochemical characterizations showed that p52ntk and p56ntk were localized to the cytoplasm, and that they partially accumulated in the detergent-insoluble cellular fraction. This last finding suggested that the Ntk proteins can associate with the cytoskeleton. Finally, through linkage analysis of two multilocus crosses, the ntk gene was mapped to Chromosome 10 in the mouse. Taken together, these data showed that ntk, a csk-related tyrosine protein kinase gene, encodes two protein isoforms expressed in distinct cell types. Moreover, they raised the possibility that Ntk may be involved in the regulation of Src-like enzymes in detergent-insoluble cellular compartments.
最近，我们和其他研究人员克隆了编码抑制性酪氨酸蛋白激酶Csk家族第二个成员的cDNA，我们将其命名为Ntk。有趣的是，两个独立研究小组发表的小鼠ntk cDNA序列在其5'端附近存在或不存在一个136个核苷酸的插入片段上有所不同。在本报告中，我们证明这个136个核苷酸的序列可能对应于ntk基因中的一个完整外显子（称为外显子2），并且这两种类型的cDNA/转录本是通过可变剪接产生的。使用核糖核酸酶保护试验还确定，脑和淋巴器官以及大多数造血细胞主要表达缺乏外显子2的ntk转录本。相反，选定的造血细胞系，如未成熟髓样细胞系32D cl3(G)和WEHI-3B，只含有带有外显子2的RNA。有趣的是，外显子2在ntk转录本中引入了一个新的框内上游AUG，其处于适合翻译起始的环境中。有证据表明这个AUG在体内被利用，并且它将Ntk的氨基末端序列延长了40个氨基酸。实际上，虽然缺乏外显子2的ntk RNA被翻译成一种52千道尔顿（kDa）的多肽（p52ntk），但那些带有外显子2的RNA产生了一种56 kDa的蛋白质（p56ntk）。此外，针对外显子2编码的新氨基末端序列的抗血清识别p56ntk，而不识别p52ntk。进一步的生化特性表明，p52ntk和p56ntk定位于细胞质，并且它们部分积累在去污剂不溶性细胞组分中。这一最新发现表明Ntk蛋白可以与细胞骨架结合。最后，通过对两个多位点杂交的连锁分析，将ntk基因定位到小鼠的10号染色体上。综上所述，这些数据表明ntk，一个与csk相关的酪氨酸蛋白激酶基因，编码在不同细胞类型中表达的两种蛋白质异构体。此外，它们还提出了Ntk可能参与去污剂不溶性细胞区室中Src样酶调节的可能性。
