Intracellular interaction of myosin light chain kinase with macrophage migration inhibition factor (MIF) in endothelium

Raj Wadgaonkar, Steven M. Dudek, Ari L. Zaiman, Laura Linz-McGillem, Alexander D. Verin, Saule Nurmukhambetova, Lewis H. Romer, Joe G.N. Garcia

Research output: Contribution to journalArticle

Abstract

The endothelial cell Ca2+/calmodulin (CaM)-dependent myosin light chain kinase isoform (ECMLCK) is a multifunctional contractile effector involved in vascular barrier regulation, leukocyte diapedesis, apoptosis, and angiogenesis. The EC MLCK isoform and its splice variants contain a unique N-terminal sequence not present in the smooth muscle MLCK isoform (SM MLCK), which allows novel upregulation of MLCK activation by signaling cascades including p60src. The yeast two-hybrid assay system using the entire EC MLCK1 N-terminus (922 aa) as bait, identified additional stable MLCK binding partners including the 12 KDa macrophage migration inhibitory factor (MIF). This finding was confirmed by cross immunoprecipitation assays under non-denaturing conditions and by GST pu 11 down experiments using GST-N-terminal MLCK (#1-923) and MLCK N-terminal deletion mutants in TNFα- and thrombin-stimulated endothelium. This EC MLCK-MIF interaction was shown biochemically and by immunofluorescent microscopy to be enhanced in TNFα- and thrombin-stimulated endothelium, both of which induce increased MLCK activity. Thrombin induced the colocalization of an epitope-tagged, full-length MIF fusion protein with phosphorylated MLC along peripheral actin stress fibers. Together these studies suggest that the novel interaction between MIF and MLCK may have important implications for the regulation of both non-muscle cytoskeletal dynamics as well as pathobiologic vascular events that involve MLCK.

Original languageEnglish (US)
Pages (from-to)849-858
Number of pages10
JournalJournal of cellular biochemistry
Volume95
Issue number4
DOIs
StatePublished - Jul 1 2005

Keywords

  • Actin
  • Cytoskeleton
  • TNF
  • Thrombin
  • Yeast two-hybrid screening

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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  • Cite this

    Wadgaonkar, R., Dudek, S. M., Zaiman, A. L., Linz-McGillem, L., Verin, A. D., Nurmukhambetova, S., Romer, L. H., & Garcia, J. G. N. (2005). Intracellular interaction of myosin light chain kinase with macrophage migration inhibition factor (MIF) in endothelium. Journal of cellular biochemistry, 95(4), 849-858. https://doi.org/10.1002/jcb.20472