Microfilament-binding properties of N-terminal extension of the isoform of smooth muscle long myosin light chain kinase.
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.
Yang
Chun Xiang
CX
Model Animal Research Center and National Key Lab of Medicine, Nanjing University, Nanjing 210061, China.
Chen
Hua Qun
HQ
Chen
Chen
C
Yu
Wei Ping
WP
Zhang
Wen Cheng
WC
Peng
Ya Jin
YJ
He
Wei Qi
WQ
Wei
Dong Mei
DM
Gao
Xiang
X
Zhu
Min Sheng
MS
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
D000199:Actins; D007527:Isoenzymes; D008840:Microfilament Proteins; D011994:Recombinant Proteins; C052523:actin filament bundling proteins; D009219:Myosin-Light-Chain Kinase