title: Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase.
abstract: Myosin light chain kinases (MLCK) phosphorylate the regulatory light chain of myosin II in thick filaments and bind to F-actin-containing thin filaments with high affinity. The ability of short myosin light chain kinase (S-MLCK) to bind F-actin is structurally attributed to the DFRXXL regions in its N-terminus. The long myosin light chain kinase (L-MLCK) has two additional DFRXXL motifs and six Ig-like modules in its N-terminal extension. The six Ig-like modules are capable of binding to stress fibers independently. Our results from the imaging analysis demonstrated that the first two intact Ig-like modules (2Ig) in N-terminal extension of L-MLCK is the minimal binding module required for microfilament binding. Binding assay confirmed that F-actin was able to bind 2Ig. Stoichiometries of 2Ig peptide were similar for myofilament or pure F-actin. The binding affinities were slightly lower than 5DFRXXL peptide as reported previously. Similar to DFRXXL peptides, the 2Ig peptide also caused efficient F-actin bundle formation in vitro. In the living cell, over-expression of 2Ig fragment increased "spike"-like protrusion formation with over-bundled F-actin. Our results suggest that L-MLCK may act as a potent F-actin bundling protein via its DFRXXL region and the 2Ig region, implying that L-MLCK plays a role in cytoskeleton organization.
authors.affiliation: Model Animal Research Center and National Key Lab of Medicine, Nanjing University, Nanjing 210061, China.
authors.forename: Chun Xiang
authors.initials: CX
authors.lastname: Yang
authors.forename: Hua Qun
authors.initials: HQ
authors.lastname: Chen
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.forename: Wei Ping
authors.initials: WP
authors.lastname: Yu
authors.forename: Wen Cheng
authors.initials: WC
authors.lastname: Zhang
authors.forename: Ya Jin
authors.initials: YJ
authors.lastname: Peng
authors.forename: Wei Qi
authors.initials: WQ
authors.lastname: He
authors.forename: Dong Mei
authors.initials: DM
authors.lastname: Wei
authors.forename: Xiang
authors.initials: X
authors.lastname: Gao
authors.forename: Min Sheng
authors.initials: MS
authors.lastname: Zhu
mesh_terms: D000199:Actins / Q000378:metabolism; D020816:Amino Acid Motifs; D000818:Animals; D019556:COS Cells; D022081:Cell Surface Extensions / Q000648:ultrastructure; D002645:Chickens; D002522:Chlorocebus aethiops; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism* / Q000648:ultrastructure; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D018482:Muscle, Skeletal; D009130:Muscle, Smooth / Q000201:enzymology*; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D041681:NIH 3T3 Cells / Q000276:immunology / Q000648:ultrastructure; D011485:Protein Binding; D011554:Pseudopodia / Q000648:ultrastructure; D011994:Recombinant Proteins / Q000235:genetics
chemical_list: D000199:Actins; D007527:Isoenzymes; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C052523:actin filament bundling proteins; D009219:Myosin-Light-Chain Kinase