Cell biochemistry
Novel ZIP kinase isoform lacks leucine zipper

https://doi.org/10.1016/j.abb.2006.09.026Get rights and content

Abstract

Zipper-interacting protein kinase (ZIP kinase) has been thought to be involved in apoptosis and the C-terminal leucine zipper motif is important for its function. Recent studies have revealed that ZIP kinase also plays a role in regulating myosin phosphorylation. Here, we found novel ZIP kinase isoform in which the C-terminal non-kinase domain containing a leucine zipper is eliminated (hZIPK-S). hZIPK-S binds to myosin phosphatase targeting subunit 1(MYPT1) similar to the long isoform (hZIPK-L). In addition, we found that hZIPK-S as well as hZIPK-L bind to myosin. These results indicate that a leucine zipper is not critical for the binding of ZIP kinase to MYPT1 and myosin. Consistently, hZIPK-S localized with stress-fibers where they co-localized with myosin. The residues 278–311, the C-terminal side of the kinase domain common to the both isoforms, is involved in the binding to MYPT1, while the myosin binding domain is within the kinase domain. These results suggest that the newly found hZIPK-S as well as the long isoform play an important role in the regulation of myosin phosphorylation.

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.).

References (37)

  • M. Murata-Hori et al.

    FEBS Lett.

    (1999)
  • N. Niiro et al.

    J. Biol. Chem.

    (2001)
  • M. Ikebe et al.

    J. Biol. Chem.

    (1985)
  • M. Ikebe et al.

    J. Biol. Chem.

    (1986)
  • J.C. Colburn et al.

    J. Biol. Chem.

    (1988)
  • K. Itoh et al.

    Biochim. Biophys. Acta

    (1992)
  • O.H. Choi et al.

    J. Biol. Chem.

    (1994)
  • J. Feng et al.

    J. Biol. Chem.

    (1999)
  • M. Ikebe et al.

    J. Biol. Chem.

    (1985)
  • M. Ikebe et al.

    J. Biol. Chem.

    (1994)
  • J.A. Spudich et al.

    J. Biol. Chem.

    (1971)
  • M. Ikebe et al.

    J. Biol. Chem.

    (2001)
  • P.R. Graves et al.

    J. Biol. Chem.

    (2005)
  • P.K. Grewal et al.

    Gene

    (1999)
  • A. Endo et al.

    J. Biol. Chem.

    (2004)
  • N. Sato et al.

    Immunol. Lett.

    (2006)
  • T. Kawai et al.

    Mol. Cell Biol.

    (1998)
  • D. Kogel et al.

    Oncogene

    (1998)
  • Cited by (7)

    View all citing articles on Scopus
    View full text