Real-time imaging of myosin II regulatory light-chain phosphorylation using a new protein biosensor.
Phosphorylation of the RMLC (regulatory myosin light chain) regulates the activity of myosin II, which is critically involved in the motility of both muscle and non-muscle cells. There are both Ca2+-dependent and -independent pathways for RMLC phosphorylation in smooth-muscle cells, and the latter pathway is often involved in an abnormal contractility in pathological states such as asthma and hypertension. Therefore pharmacological interventions of RMLC phosphorylation may have a therapeutic value. In the present study, we developed a new genetically encoded biosensor, termed CRCit (ECFP-RMLC-Citrine, where ECFP is enhanced cyan fluorescent protein), that detects RMLC phosphorylation using fluorescence resonance energy transfer between two variants of the green fluorescent protein fused to both the N- and C-termini of RMLC. When expressed in primary cultured vascular smooth-muscle cells, CRCit detected the Ca2+-dependent RMLC phosphorylation with a high spatiotemporal resolution. Furthermore, we could specifically assay the agonist-induced Ca2+-independent phosphorylation of RMLC when Ca2+ signalling in cells expressing CRCit was suppressed. Thus CRCit may also be used for the high throughput screening of compounds that inhibit abnormal smooth-muscle contraction.
Yamada
Aki
A
Department of Pharmacology, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo, 113-0033, Japan.
Hirose
Kenzo
K
Hashimoto
Akiko
A
Iino
Masamitsu
M
D000818:Animals; D001011:Aorta / Q000737:chemistry / Q000378:metabolism; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D015374:Biosensing Techniques; D002478:Cells, Cultured; D003199:Computer Systems; D004789:Enzyme Activation / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology; D009154:Mutation / Q000235:genetics; D032389:Myocytes, Smooth Muscle / Q000737:chemistry / Q000378:metabolism; D018994:Myosin Light Chains / Q000235:genetics / Q000378:metabolism*; D009219:Myosin-Light-Chain Kinase; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D054460:rho-Associated Kinases
D001426:Bacterial Proteins; C485182:Cyan Fluorescent Protein; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D018994:Myosin Light Chains; C434479:citrine protein, bacteria; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D054460:rho-Associated Kinases; D009219:Myosin-Light-Chain Kinase