Myosin Light Chain Kinase‐210 Induces ER‐PM Junctions and STIM1 Puncta Formation to Augment Store‐Operated Ca2+ Entry

Abstract

Myosin light chain kinase‐Long (MYLK‐L or MYLK‐210), a Ca2+‐calmodulin dependent kinase containing a unique 922‐amino acid N‐terminal domain is a predominant isoform of MYLK expressed in endothelial cells (ECs). N‐terminus of MYLK‐210 has been shown to contain several single nucleotide polymorphisms (SNPs) in an actin binding region which is associated with severity of acute lung injury (ALI). However, significance of SNPs in regulating MYLK‐210 function remains unclear. The classical function of MYLK‐210 is to catalyze the phosphorylation of myosin light chain to induce EC contraction, leading to inter‐endothelial gap formation and increase permeability, a hall mark of ALI. Intriguingly, we showed that MYLK‐210, through its actin‐binding motif, regulates store‐operated Ca2+ entry (SOCE) but not receptor‐operated Ca2+ entry (ROCE). We showed that MYLK‐210 null endothelial cells (ECs) or MYLK‐210 depleted ECs displayed diminished Ca2+ entry in response to SOCE‐activator thapsigargin and thrombin. We generated MYLK‐210 constructs lacking C‐terminus (N‐MYLK) or N‐terminus (C‐MYLK) to investigate the role of specific domains of MYLK‐210 in inducing SOCE activity. We found that N‐MYLK but not C‐MYLK rescued SOCE activity in MYLK‐210 null ECs, indicating thereby that MYLK regulates SOCE in a kinase independent manner. Additionally, SOCE activation failed to increase lung vascular hyper‐permeability in MYLK‐210 null mice. We identified unique actin binding motif, DVRGLL within N terminus of MYLK‐L which enables MYLK‐210 to induce SOCE. Stromal‐interaction molecule 1 (STIM1), upon sensing the depletion of (Ca2+) from the endoplasmic reticulum (ER) store, organizes as puncta which translocate to plasma membrane that trigger SOCE via plasmalemmal Ca2+selective channels such as transient receptor potential channel 1 (TRPC1) and Orai1. Thus, we addressed if MYLK‐L controlled STIM1 function by regulating puncta formation. We showed that STIM1 formed puncta in control ECs but these puncta were markedly reduced in MYLK depleted ECs. Formation of endoplasmic reticulum (ER)‐plasma membrane (PM) junction is required for STIM1 activation of SOCE. Using MAPPER (membrane‐attached peripheral ER) that selectively labels ER–PM junctions, we found that MYLK induced ER‐PM junction, but these junctions were markedly reduced in the density in MYLK‐knockdown cells. Our study thereby defines for the first time the clinical validity of SNPs within actin binding region of MYLK in inducing ALI through organizing ER‐PM junctions, STIM1 activity, SOCE and lung vascular hyperpermeability.Support or Funding InformationNational Institute of Health (NIH)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.