Mutation Analysis of the Non-Muscle Myosin Light Chain Kinase (MLCK) Deletion Constructs on CV1 Fibroblast Contractile Activity and Proliferation

J Cell Biochem. 2003 Feb 15;88(3):623-34. doi: 10.1002/jcb.10370.

Abstract

Smooth muscle myosin light chain kinase (MLCK) is a multifunctional molecule composed of an N-terminal actin binding domain, a central kinase domain, and C-terminal calmodulin- and myosin-binding domains. We previously cloned and characterized a novel MLCK isoform from endothelial cells (EC MLCK) consisting of 1,914 amino acids displaying a higher molecular weight (210 kDa) and a novel-amino-terminal stretch of 922 amino acids not shared by the smooth muscle isoform (smMLCK, 150 kDa). To further define the role of specific EC MLCK motifs in endothelial and non-muscle cells, we constructed two epitope-tagged EC MLCK deletion mutants in mammalian expression vectors lacking either the C-terminal auto-inhibitory and calmodulin-binding domain (EC MLCK1745) or the ATP-binding site (EC MLCKATPdel). Expression of EC MLCK1745 in CV1 fibroblasts showed increased basal actin stress fiber formation, which was markedly enhanced after tumor necrosis factor (TNF-alpha) or thrombin treatment. Distribution of EC MLCK1745 was largely confined to stress fibers, cortical actin filaments, and focal adhesion contacts, and co-localized with myosin light chains (MLCs) diphosphorylated on Ser(19) and Thr(18). In contrast, immunofluorescence staining demonstrated that EC MLCKATPdel abolished thrombin- and TNFalpha-induced stress fiber formation and MLC phosphorylation, suggesting this kinase-dead mutant functions as a dominant-negative MLCK construct, thereby confirming the role of EC MLCK in stress fiber formation. Finally, we compared the serum-stimulated growth rate of mutant MLCK-transfected fibroblasts to sham controls, and found EC MLCK1745 to augment thymidine incorporation whereas EC MLCKATPdel reduced CV1 growth rates. These data demonstrate the necessary role for MLCK in driving the contractile apparatus via MLC phosphorylation, which can alter fibroblast growth and contractility.

MeSH terms

  • Animals
  • Calmodulin / antagonists & inhibitors
  • Calmodulin / metabolism
  • Cell Division / physiology*
  • Cell Line
  • Fibroblasts / cytology
  • Fibroblasts / metabolism*
  • Humans
  • Muscle Contraction / physiology*
  • Mutation*
  • Myosin-Light-Chain Kinase / genetics*
  • Myosin-Light-Chain Kinase / metabolism*
  • Phosphorylation
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Stress Fibers / metabolism
  • Thrombin / metabolism

Substances

  • Calmodulin
  • Recombinant Fusion Proteins
  • Myosin-Light-Chain Kinase
  • Thrombin