Abl tyrosine kinase phosphorylates nonmuscle Myosin light chain kinase to regulate endothelial barrier function.
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.
Dudek
Steven M
SM
Institute for Personalized Respiratory Medicine, Section of Pulmonary, Critical Care, Sleep, and Allergy, University of Illinois at Chicago, Chicago, IL 60612, USA.
Chiang
Eddie T
ET
Camp
Sara M
SM
Guo
Yurong
Y
Zhao
Jing
J
Brown
Mary E
ME
Singleton
Patrick A
PA
Wang
Lichun
L
Desai
Anjali
A
Arce
Fernando T
FT
Lal
Ratnesh
R
Van Eyk
Jennifer E
JE
Imam
Syed Z
SZ
Garcia
Joe G N
JG
D000199:Actins / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002199:Capillary Permeability / Q000187:drug effects; D022461:Caveolins / Q000378:metabolism; D002460:Cell Line; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism; D042783:Endothelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004789:Enzyme Activation; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology; D013058:Mass Spectrometry; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism; D018625:Microscopy, Atomic Force; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D016315:Proto-Oncogene Proteins c-abl / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D000199:Actins; D022461:Caveolins; D008246:Lysophospholipids; D049452:Green Fluorescent Proteins; C060506:sphingosine 1-phosphate; D014443:Tyrosine; D016315:Proto-Oncogene Proteins c-abl; D009219:Myosin-Light-Chain Kinase; D013110:Sphingosine