Abl tyrosine kinase phosphorylates nonmuscle myosin light chain kinase to regulate endothelial barrier function

Steven M. Dudek, Eddie T. Chiang, Sara M. Camp, Yurong Guo, Jing Zhao, Mary E. Brown, Patrick A. Singleton, Lichun Wang, Anjali Desai, Fernando T. Arce, Ratnesh Lal, Jennifer E. Van Eyk, Syed Z. Imam, Joe G.N. Garcia

Research output: Contribution to journalArticle

65 Scopus citations

Abstract

Nonmuscle myosin light chain kinase (nmMLCK), a multi-functional cytoskeletal protein critical to vascular homeostasis, is highly regulated by tyrosine phosphorylation. We identified multiple novel c-Abl-mediated nmMLCK phosphorylation sites by mass spectroscopy analysis (including Y231, Y464, Y556, Y846) and examined their influence on nmMLCK function and human lung endothelial cell (EC) barrier regulation. Tyrosine phosphorylation of nmMLCK increased kinase activity, reversed nmMLCK-mediated inhibition of Arp2/3-mediated actin polymerization, and enhanced binding to the critical actin-binding phosphotyrosine protein, cortactin. EC challenge with sphingosine 1-phosphate (S1P), a potent barrier-enhancing agonist, resulted in c-Abl and phosphorylated nmMLCK recruitment into caveolin-enriched microdomains, rapid increases in Abl kinase activity, and spatial targeting of c-Abl to barrier-promoting cortical actin structures. Conversely, reduced c-Abl expression in EC (siRNA) markedly attenuated S1P-mediated cortical actin formation, reduced the EC modulus of elasticity (assessed by atomic force microscopy), reduced nmMLCK and cortactin tyrosine phosphorylation, and attenuated S1P-mediated barrier enhancement. These studies indicate an essential role for Abl kinase in vascular barrier regulation via posttranslational modification of nmMLCK and strongly support c-Abl-cortactin-nmMLCK interaction as a novel determinant of cortical actin-based cytoskeletal rearrangement critical to S1P-mediated EC barrier enhancement.

Original languageEnglish (US)
Pages (from-to)4042-4056
Number of pages15
JournalMolecular biology of the cell
Volume21
Issue number22
DOIs
StatePublished - Nov 15 2010

ASJC Scopus subject areas

  • Molecular Biology
  • Cell Biology

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    Dudek, S. M., Chiang, E. T., Camp, S. M., Guo, Y., Zhao, J., Brown, M. E., Singleton, P. A., Wang, L., Desai, A., Arce, F. T., Lal, R., Van Eyk, J. E., Imam, S. Z., & Garcia, J. G. N. (2010). Abl tyrosine kinase phosphorylates nonmuscle myosin light chain kinase to regulate endothelial barrier function. Molecular biology of the cell, 21(22), 4042-4056. https://doi.org/10.1091/mbc.E09-10-0876