1. ¹Model Animal Research Center and National Key Lab of Medicine, Nanjing University, Nanjing 210061, China
2. ²College of Life Science, Nanjing Normal University, Nanjing 210097, China
3. ³Medical College, Southeast University, Nanjing 210096, China
4. ⁴Huadong Research Institute for Medical Biotechnics, Nanjing 210002, China

Correspondence: Min Sheng Zhu, Tel: 86-25-58641529; Fax: 86-25-58641500; E-mail: zms@nicemice.cn

^*These authors contributed equally to this work.

Received 26 September 2005; Revised 10 January 2006; Accepted 11 January 2006.

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.