Calmodulin binding domains: characterization of a phosphorylation and calmodulin binding site from myosin light chain kinase

Biochemistry ◽  
1986 ◽  
Vol 25 (6) ◽  
pp. 1458-1464 ◽  
Author(s):  
Thomas J. Lukas ◽  
Wilson H. Burgess ◽  
Franklyn G. Prendergast ◽  
Wai Lau ◽  
D. Martin Watterson
Keyword(s):  
Binding Site ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Calmodulin Binding ◽  
Binding Domains

scholarly journals Serine/threonine phosphorylation of calmodulin modulates its interaction with the binding domains of target enzymes

1999 ◽  
Vol 344 (2) ◽  
pp. 403-411 ◽  
Author(s):  
Estelle LECLERC ◽  
Chantal CORTI ◽  
Holger SCHMID ◽  
Stefan VETTER ◽  
Peter JAMES ◽  
...  
Keyword(s):  
Dissociation Constant ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Camp Phosphodiesterase ◽  
Calmodulin Binding ◽  
Binding Domains ◽  
Order Of Magnitude ◽  
Oxide Synthase ◽  
Muscle Myosin

The interaction of serine/threonine-phosphorylated calmodulin with synthetic peptides corresponding to the calmodulin-binding domains of six enzymes has been studied by fluorescence spectroscopy. For five peptides, the dissociation constant of the calmodulin-peptide complex (Kd) increased when calmodulin was phosphorylated. An increase of more than one order of magnitude was observed with peptides derived from smooth-muscle myosin light-chain kinase and cAMP phosphodiesterase. In contrast, only a slight increase in Kd was noted with two peptides derived from the plasma membrane Ca2+-ATPase and for the peptide derived from nitric oxide synthase. No significant change in affinity was detected with the peptide derived from calcineurin. In contrast, a decrease in the dissociation constant was observed with the peptide derived from the Ca2+-calmodulin dependent kinase II. Phosphorylation also affected the peptide-calmodulin binding stoichiometry: a decrease from two to one binding sites was observed with the peptides derived from myosin light-chain kinase and phosphodiesterase.

scholarly journals Affinity labelling of smooth-muscle myosin light-chain kinase with 5′-[p-(fluorosulphonyl)benzoyl]adenosine

1993 ◽  
Vol 296 (1) ◽  
pp. 53-58 ◽  
Author(s):  
H Komatsu ◽  
M Ikebe
Keyword(s):  
Smooth Muscle ◽  
Binding Site ◽  
Light Chain ◽  
Myosin Light Chain Kinase ◽  
Myosin Light Chain ◽  
Smooth Muscle Myosin ◽  
Atp Binding ◽  
Atp Binding Site ◽  
Calmodulin Binding ◽  
Muscle Myosin

5′-(p-(Fluorosulphonyl)[14C]benzoyl)adenosine (FSBA) was synthesized and used as a probe to study the ATP-binding site of smooth-muscle myosin light-chain kinase (MLCK). FSBA modified both free MLCK and calmodulin/MLCK complex, resulting in inactivation of the kinase activity. Nearly complete protection of the calmodulin/MLCK complex against FSBA modification was obtained by addition of excess ATP whereas MLCK activity alone was lost in a dose-dependent manner even in the presence of excess ATP. These results suggest that FSBA modified ATP-binding sites and ATP-independent sites, and the latter sites are protected by calmodulin binding. The results also suggest that the ATP-binding site is accessible to the nucleotide substrate regardless of calmodulin binding. The FSBA-labelled MLCK was completely proteolysed by alpha-chymotrypsin, and the 14C-labelled peptides were isolated and sequenced. The sequence of the labelled peptide was Ala-Gly-X-Phe, where X is the labelled residue. The sequence was compared with the known MLCK sequence, and the labelled residue was identified as lysine-548, which is located downstream of the GXGXXG motif conserved among ATP-utilizing enzymes.

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