Komatsu, S. & Ikebe, M. ZIP kinase is responsible for the phosphorylation of myosin II and necessary for cell motility in mammalian fibroblasts. J. Cell Biol. 165, 243-254

Department of Physiology, University of Massachusetts Medical School, Worcester, MA 01655
The Journal of Cell Biology (Impact Factor: 9.83). 05/2004; 165(2):243-54. DOI: 10.1083/jcb.200309056
Source: PubMed


Reorganization of actomyosin is an essential process for cell migration and myosin regulatory light chain (MLC20) phosphorylation plays a key role in this process. Here, we found that zipper-interacting protein (ZIP) kinase plays a predominant role in myosin II phosphorylation in mammalian fibroblasts. Using two phosphorylation site-specific antibodies, we demonstrated that a significant portion of the phosphorylated MLC20 is diphosphorylated and that the localization of mono- and diphosphorylated myosin is different from each other. The kinase responsible for the phosphorylation was ZIP kinase because (a) the kinase in the cell extracts phosphorylated Ser19 and Thr18 of MLC20 with similar potency; (b) immunodepletion of ZIP kinase from the cell extracts markedly diminished its myosin II kinase activity; and (c) disruption of ZIP kinase expression by RNA interference diminished myosin phosphorylation, and resulted in the defect of cell polarity and migration efficiency. These results suggest that ZIP kinase is critical for myosin phosphorylation and necessary for cell motile processes in mammalian fibroblasts.

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Available from: Satoshi Komatsu, Jul 15, 2014
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    • "Zipper-interacting protein kinase ((ZIPK), also known as DAPK3 or Dlk) [56] belongs to the family of death-associated protein kinases (DAPK) [57,58]. ZIPK controls a variety of cell processes, including cell motility [59] and smooth muscle contraction [12,60,61]. Identified in 1998 [62,63], ZIPK possesses an amino-terminal kinase domain, a putative central autoinhibitory domain and a carboxyl-terminal leucine zipper motif that permits dimerization and interactions with other proteins (Figure 2). "
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    ABSTRACT: Smooth muscle is a major component of most hollow organ systems (e.g., airways, vasculature, bladder and gut/gastrointestine); therefore, the coordinated regulation of contraction is a key property of smooth muscle. When smooth muscle functions normally, it contributes to general health and wellness, but its dysfunction is associated with morbidity and mortality. Rho-associated protein kinase (ROCK) is central to calcium-independent, actomyosin-mediated contractile force generation in the vasculature, thereby playing a role in smooth muscle contraction, cell motility and adhesion. Recent evidence supports an important role for ROCK in the increased vasoconstriction and remodeling observed in various models of hypertension. This review will provide a commentary on the development of specific ROCK inhibitors and their clinical application. Fasudil will be discussed as an example of bench-to-bedside development of a clinical therapeutic that is used to treat conditions of vascular hypercontractility. Due to the wide spectrum of biological processes regulated by ROCK, many additional clinical indications might also benefit from ROCK inhibition. Apart from the importance of ROCK in smooth muscle contraction, a variety of other protein kinases are known to play similar roles in regulating contractile force. The zipper-interacting protein kinase (ZIPK) and integrin-linked kinase (ILK) are two well-described regulators of contraction. The relative contribution of each kinase to contraction depends on the muscle bed as well as hormonal and neuronal stimulation. Unfortunately, specific inhibitors for ZIPK and ILK are still in the development phase, but the success of fasudil suggests that inhibitors for these other kinases may also have valuable clinical applications. Notably, the directed inhibition of ZIPK with a pseudosubstrate molecule shows unexpected effects on the contractility of gastrointestinal smooth muscle.
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    • "Dlk possesses a highly homologous kinase domain (with 79.5% identity to that of DAPk) in the N-terminus, a rather short C-terminus which contains several nuclear localization signal motifs and a leucine zipper domain, without either death domain or CaM regulatory domain of DAPk. Studies demonstrated that Dlk could autophosphorylate itself and phosphorylate a variety of substrates including myosin light chain (MLC) and core histones in vitro [17] [18] [19]. The C-terminal leucine zipper domain of Dlk mediates its homodimerization and its interactions with other leucine zipper-containing proteins including AATF, par-4 and CDC5 [20] [21] [22]. "
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    ABSTRACT: Death-associated protein kinase (DAPk) family has emerged as a novel subfamily of pro-apoptotic serine/threonine kinase in the last 10 years. Although the functions of DAPk have been well documented, those of other family members remain uncertain. In this work, we characterized the expression pattern of human DAPk like kinase/Zipper interacting protein kinase (Dlk/ZIP kinase) in cancer specimens and cell lines. Dlk expression level was significantly down-regulated in cervical carcinoma cells compared to the surrounding non-tumorous tissues. Overexpression of Dlk led to cell morphological changes, suppressed colony formation and elevated cell apoptosis in cancer cell lines. Both the kinase activity and the cytoplasmic localization were required for its pro-apoptotic tendency. Mechanism exploration revealed that upon serum deprivation, Dlk overexpression could sensitize cells to apoptosis while overexpression of the kinase inactive mutant (Dlk-K42A) was able to rescue apoptotic cell death. Our data thus implicates that Dlk plays a positive role in modulating death-related signaling pathways. Reconstitution of Dlk expression might bring a potential therapeutic approach to cervical carcinoma treatments.
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    • "The bisphosphorylation of MLC can also occur in a Ca 2+ -independent manner, through a pathway involving the AGC (containing PKA, PKG, and PKC families ) Ser/Thr kinase family member Rho-kinase (ROCK). ROCK activity enhances MLC phosphorylation in a dual way by increasing its bisphosphorylation directly [3] [4] and at the same time by preventing its dephosphorylation through inhibition of the myosin light chain phosphatase [5] [6]. Inhibition of MLC bisphosphorylation through ROCK inhibition has been shown to result phenotypically in the loss of stress fibers [7] [8], cellular relaxation [8] [9], and the prevention of agonist-induced neurite retraction [3] [10] [11]. "
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    ABSTRACT: Here, we describe the first example of a cell-based myosin light chain phosphorylation assay in 96-well format that allows for the rapid screening of novel Rho-kinase inhibitors. We obtained IC(50) values for the prototypic Rho-kinase inhibitors Y-27632 (1.2+/-0.05microM) and Fasudil (3.7+/-1.2microM) that were similar to those previously published utilizing electrophoresis-based methodologies. H-1152P, a Fasudil analog showed an IC(50) value of 77+/-30nM. Data derived from a set of 21 novel Rho-kinase inhibitors correlate with those generated by a well-established cell-based phenotypic Rho-kinase inhibition assay (R(2)=0.744). These results show that imaging technology measuring changes in myosin light chain phosphorylation can be used to rapidly generate quantitative IC(50) values and to screen a larger set of small molecule Rho-kinase inhibitors and suggests that this approach can be broadly applied to other cell lines and signaling pathways.
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