2 DISTINCT PROTEIN ISOFORMS ARE ENCODED BY NTK, A CSK-RELATED TYROSINE PROTEIN-KINASE GENE

Recently, we and others have cloned cDNAs encoding a second member of the Csk family of inhibitory tyrosine protein kinases, which we have t ermed Ntk. Intriguingly, the mouse ntk cDNA sequences published by two independent groups differed by the presence or absence of a 136 nucle otide-insert near their 5' ends. In this report, we demonstrate that t his 136 nucleotide-sequence likely corresponds to a complete exon in t he ntk gene (termed exon 2), and that the two types of cDNAs/transcrip ts are produced by alternative splicing. Using ribonuclease protection assays, it was also established that brain and lymphoid organs, as we ll 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 po ssessed exon 2-bearing RNAs. Interestingly, exon 2 introduced a novel in-frame upstream AUG in the ntk transcript, which is in the appropria te context for translation initiation. Evidence was obtained that this AUG is utilized in vivo, and that it extends the amino-terminal seque nce of Ntk by 40 amino acids. Indeed, while exon 2-deficient ntk RNAs were translated into a 52 kilodalton (kDa) polypeptide (p52(ntk)), tho se bearing exon 2 produced a 56 kDa protein (p56(ntk)). Furthermore, p 56(ntk), but not p52(ntk), was recognized by an antiserum directed aga inst the novel amino-terminal sequence encoded by exon 2. Additional b iochemical characterizations showed that p52(ntk) and p56(ntk) were lo calized to the cytoplasm, and that they partially accumulated in the d etergent-insoluble cellular fraction. This last finding suggested that the Ntk proteins can associate with the cytoskeleton. Finally, throug h 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 i soforms expressed in distinct cell types. Moreover, they raised the po ssibility that Ntk may be involved in the regulation of Src-Like enzym es in detergent-insoluble cellular compartments.