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 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 7.70 +/- 1.86% of extensor digitorum longus myofibrils displayed aggregates with a 36.44% reduction in myofibril diameter, whereas 65.13 +/- 3.42% of diaphragm myofibrils displayed aggregates and the myofibril diameter was reduced by 43.08%. Electron microscopy examination suggested that the aggregates were deposited at the mitochondria, resulting in structural impairment. As a consequence, the oxygen consumption of mitochondria in the affected muscles was also reduced. Macrophenotypic analysis showed the presence of muscular degeneration characterized by a reduction in force development, faster fatigue, decreased myofibril diameters, and structural alterations. In summary, our study revealed the existence of a novel aggregation domain in L-MLCK and provided a direct link between L-MLCK and aggregation. The possible significance and mechanism underlying the aggregation-based pathological processes mediated by L-MLCK are also discussed.
Zhang
Wen-Cheng
WC
Model Animal Research Center, Nanjing University, China.
Peng
Ya-Jing
YJ
He
Wei-Qi
WQ
Lv
Ning
N
Chen
Chen
C
Zhi
Gang
G
Chen
Hua-Qun
HQ
Zhu
Min-Sheng
MS
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002645:Chickens; D003964:Diaphragm / Q000166:cytology / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D008969:Molecular Sequence Data; D009119:Muscle Contraction; D018763:Muscle Fatigue; D015879:Myosin Subfragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020539:Sequence Analysis, Protein
D015879:Myosin Subfragments; D011993:Recombinant Fusion Proteins; D009219:Myosin-Light-Chain Kinase