Phosphorylation of caldesmon by myosin light chain kinase increases its binding affinity for phosphorylated myosin filaments.
Phosphorylation of myosin by myosin light chain kinase (MLCK) is essential for smooth muscle contraction. In this study we show that caldesmon (CaD) is also phosphorylated in vitro by MLCK. The phosphorylation is calcium- and calmodulin (CaM)-dependent and requires a MLCK concentration close to that found in vivo. On average, approximately 2 mol P(i) per mol of CaD are incorporated at Thr-626 and Thr-693, with additional partial phosphorylation at Ser-658 and Ser-702. The phosphorylation rate for CaD is 20- to 50-fold slower than that for filamentous myosin; faster relative rates were obtained with CaD added to purified actomyosin or myosin preparations containing endogenous MLCK/CaM complex. Addition of CaM also augmented CaD phosphorylation. We further demonstrate that [³²P] labeled CaD binds much more readily to phosphorylated filamentous myosin than to unphosphorylated myosin. For actomyosin, CaD binding affinity doubles after myosin phosphorylation, without a significant change in binding stoichiometry (approx. one CaD per myosin molecule). Unphosphorylated CaD is ineffective in competing with the phosphorylated protein for the binding site(s) on myosin filaments. The ATPase activity of reconstituted actomyosin is inhibited by unphosphorylated CaD, and this inhibition was removed by CaD phosphorylation. Our results suggest that CaD phosphorylation plays a role in modifying actomyosin interaction in vivo, particularly during prolonged muscle activation.
Institute for Biomedical Aging Research, Smooth Muscle Laboratory at the Life Science Center, Austrian Academy of Sciences, Mitterweg 24, A-6020 Innsbruck, Austria. ASobieszek@oeaw.ac.at
Apolinary
A
Sobieszek
Bettina
B
Sarg
Herbert
H
Lindner
Chun Y
CY
Seow
D000818:Animals; D001665:Binding Sites; D002148:Calmodulin-Binding Proteins / Q000378:metabolism*; D009119:Muscle Contraction / Q000502:physiology; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000378:metabolism*; D009218:Myosins / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D014422:Turkeys
D002148:Calmodulin-Binding Proteins; D010750:Phosphoproteins; D009219:Myosin-Light-Chain Kinase; D009218:Myosins