tailieunhanh - Báo cáo khoa học: Identification and functional characterization of an aggregation domain in long myosin light chain kinase

The functions of long smooth muscle myosin light chain kinase (L-MLCK), a molecule with multiple domains, are poorly understood. To examine the existence of further potentially functional domains in this molecule, we ana-lyzed its amino acid sequence with a tangoprogram and found a putative aggregation domain located at the 4Ig domain of the N-terminal extension. | ỊFEBS Journal Identification and functional characterization of an aggregation domain in long myosin light chain kinase Wen-Cheng Zhang1 Ya-Jing Peng1 Wei-Qi He1 Ning Lv2 Chen Chen1 Gang Zhi3 Hua-Qun Chen2 and Min-Sheng Zhu1 1 ModelAnimalResearch Center Nanjing University China 2 Schoolof Life Science Nanjing NormalUniversity China 3 NationalInstitute of BiologicalScience Beijing China Keywords 4Ig domain aggregation contraction mitochondria myosin light chain kinase Correspondence . Zhu ModelAnimalResearch Center of Nanjing University 12 Xue-Fu Road Pukou District Nanjing China 210061 Fax 86 2558641500 Tel 86 2558641529 E-mail zhums@ Received 14 January 2008 revised 4 March 2008 accepted 11 March 2008 doi The functions of long smooth muscle myosin light chain kinase L-MLCK a molecule with multiple domains are poorly understood. To examine the existence of further potentially functional domains in this molecule we analyzed its amino acid sequence with a TANGO program and found a putative aggregation domain located at the 4Ig domain of the N-terminal extension. To verify its aggregation capability in vitro expressible truncated L-MLCK variants driven by a cytomegalovirus promoter were transfected into cells. As anticipated only the overexpression of the 4Ig fragment led to particle formation in Colon26 cells. These particles contained 4Ig polymers and actin. Analysis with detergents demonstrated that the particles shared features in common with aggregates. Thus we conclude that the 4Ig domain has a potent aggregation ability. To further examine this aggregation domain in vivo eight transgenic mouse lines expressing the 4Ig domain 4Ig lines were generated. The results showed that the transgenic mice had typical aggregation in the thigh and diaphragm muscles. Histological examination showed that of extensor digitorum longus myofibrils displayed aggregates with a reduction in myofibril diameter whereas .