Cell biochemistryNovel ZIP kinase isoform lacks leucine zipper
Section snippets
Cloning of ZIP kinase isoforms
To clone potential splicing variants of human ZIP kinase, 1 μg total RNA from human bladder (Ambion Inc., Austin, TX) was reverse transcribed using SuperScriptII reverse transcriptase (Invitrogen Life technologies, Carlsbad, CA) and 3′-RACE adapter primer (5′-GCG AGC ACA GAA TTA ATA CGA CTC ACT ATA GG (T)12 VN-3′). 1/20 volume of reverse-transcription reaction was subjected to two rounds of polymerase chain reaction (PCR). Primer pairs used for the first round PCR were Exon2-specific forward
Molecular cloning of ZIPK isoform from human bladder
ZIP kinase contains a conserved kinase domain at the N-terminal half of the molecule. Therefore, we performed a 3′-RACE using human bladder total RNA as a template to obtain ZIP kinase isoforms because the putative ZIP kinase isoforms may contain a 3′-untranslated region different from that of known ZIP kinase (see Materials and methods). We found a cDNA fragment whose molecular mass is much smaller than the known human ZIP kinase (hZIPK-L) (Fig. 1A). This cDNA fragment was subcloned into
Discussion
We report here, a novel ZIP kinase isoform (hZIPK-S) in which a majority of originally assigned exon 8, encoding the C-terminal non-kinase domain, is spliced out. This type of splicing has been known as intron retention [31], [32]. A similar type of the splicing variants are found for myosin IXb [33] (Kambara and Ikebe, unpublished observation). Previously, MacDonald et al. [20] reported that ZIP-like kinase purified from cow bladder had an apparent molecular mass of 32 kDa that was much smaller
Acknowledgments
This work was supported by NIH Grants AR41653 and HL073050 (M.I.) and the American Heart Association Grant 0535419T (S.K.).
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