Myosin light chain kinase (MYLK) coding polymorphisms modulate human lung endothelial cell barrier responses via altered tyrosine phosphorylation, spatial localization, and lamellipodial protrusions

Abstract

Sphingosine 1-phosphate (S1P) is a potent bioactive endogenous lipid that signals a rearrangement of the actin cytoskeleton via the regulation of non-muscle myosin light chain kinase isoform (nmMLCK). S1P induces critical nmMLCK Y464 and Y471 phosphorylation resulting in translocation of nmMLCK to the periphery where spatially-directed increases in myosin light chain (MLC) phosphorylation and tension result in lamellipodia protrusion, increased cell-cell adhesion, and enhanced vascular barrier integrity. MYLK, the gene encoding nmMLCK, is a known candidate gene in lung inflammatory diseases, with coding genetic variants (Pro21His, Ser147Pro, Val261Ala) that confer risk for inflammatory lung injury and influence disease severity. The functional mechanisms by which these MYLK coding single nucleotide polymorphisms (SNPs) affect biologic processes to increase disease risk and severity remain elusive. In the current study, we utilized quantifiable cell immunofluorescence assays to determine the influence of MYLK coding SNPs on S1P-mediated nmMLCK phosphorylation and translocation to the human lung endothelial cell (EC) periphery . These disease-associated MYLK variants result in reduced levels of S1P-induced Y464 phosphorylation, a key site for nmMLCK enzymatic regulation and activation. Reduced Y464 phosphorylation resulted in attenuated nmMLCK protein translocation to the cell periphery. We further conducted EC kymographic assays which confirmed that lamellipodial protrusion in response to S1P challenge was retarded by expression of a MYLK transgene harboring the three MYLK coding SNPs. These data suggest that ARDS/severe asthma-associated MYLK SNPs functionally influence vascular barrier-regulatory cytoskeletal responses via direct alterations in the levels of nmMLCK tyrosine phosphorylation, spatial localization, and lamellipodial protrusions.

Targeting NOX4 alleviates sepsis-induced acute lung injury via attenuation of redox-sensitive activation of CaMKII/ERK1/2/MLCK and endothelial cell barrier dysfunction

“…In our study, we found a significant increase in CaMKII Thr-286 phosphorylation in CLP mice and LPS-stimulated endothelial cells, which can be inhibited by NOX4 knockdown. ERK1/2 and MLCK [ 24 , [43] , [44] , [45] , [46] ], downstream effectors of CaMKII, also exhibited increased phosphorylation in CLP mice and LPS-stimulated endothelial cells, which were significantly abrogated by NOX4 knockdown and the pharmacological inhibitor of CaMKII, KN93. Therefore, phosphorylation-dependent activation of CaMKII and its downstream effectors underlies NOX4-dependent modulation of lung permeability.…”

The Splicing Factor hnRNPA1 Regulates Alternate Splicing of the MYLK Gene

“…We have identified nonmuscle myosin light chain kinase (nmMLCK) as the principal isoform in ECs (6) and have demonstrated the role of nmMLCK in maintenance of the vascular barrier using cellular and murine models (7)(8)(9). Genetic variants in MYLK, the gene encoding the nmMLCK isoform, are associated with ARDS risk and mortality, likely via an influence, at least in part, on nmMLCK regulation of EC barrier integrity (3,(10)(11)(12)(13).…”

MYLK pathogenic variants aortic disease presentation, pregnancy risk, and characterization of pathogenic missense variants

“…These findings emphasize the fact that established functional assays do not always correlate with the pathogenicity of variants. In the long form of MLCK, which is responsible for RLC phosphorylation in non-muscle cells, the cellular localization and regulation of MLCK is dependent on phosphorylation of tyrosines in the unique N-terminal domain of the protein 15,16 . Therefore, future studies to determine how MYLK p.Tyr1575His predisposes to disease should focus on whether phosphorylation of this tyrosine plays a role in kinase activation in smooth muscle cells.…”