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Two distinct protein isoforms are encoded by ntk, a csk-related tyrosine protein kinase gene
L M Chow1, D Davidson, M Fournel
1McGill Cancer Centre, McGill University, Montréal, Canada.
Abstract:
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.
Insights
The Ntk gene produces two distinct protein isoforms, p52ntk and p56ntk, through alternative splicing involving exon 2. These isoforms are found in different cell types and may regulate Src-like enzymes in cellular compartments.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- The Csk family of inhibitory tyrosine protein kinases includes a second member, Ntk.
- Mouse ntk cDNA sequences varied, suggesting potential transcript differences.
Purpose of the Study:
- Investigate the cause of ntk cDNA sequence variations.
- Characterize the Ntk protein isoforms and their cellular localization.
- Map the ntk gene in mice.
Main Methods:
- Ribonuclease protection assays to analyze ntk transcripts.
- In vivo translation studies to identify protein products.
- Biochemical characterization of protein localization.
- Linkage analysis for gene mapping.
Main Results:
- Alternative splicing of exon 2 in the ntk gene generates two transcript types.
- Ntk transcripts lacking exon 2 are predominant in brain, lymphoid, and most hemopoietic cells.
- Exon 2-bearing transcripts are found exclusively in specific myeloid cell lines.
- Exon 2 introduces an upstream AUG, leading to a 40 amino acid N-terminal extension, producing p56ntk (vs. p52ntk without exon 2).
- p56ntk, but not p52ntk, is recognized by an antiserum against the exon 2-encoded sequence.
- Both isoforms localize to the cytoplasm, with partial accumulation in detergent-insoluble fractions, suggesting cytoskeletal association.
- The ntk gene is mapped to mouse Chromosome 10.
Conclusions:
- The ntk gene encodes two distinct protein isoforms, p52ntk and p56ntk, generated by alternative splicing.
- These isoforms exhibit differential expression patterns in various cell types.
- Ntk proteins may associate with the cytoskeleton and potentially regulate Src-like enzymes in detergent-insoluble compartments.