ADP VANADATE MEDIATED PHOTOCLEAVAGE OF MYOSIN LIGHT-CHAIN KINASE AT THE AUTOINHIBITORY REGION/

The vanadate (Vi)-mediated photocleavage reaction was used to study th e interaction between the regulatory segment and the catalytic site of smooth muscle myosin light chain kinase (MLCK), When MLCK was irradia ted with long-wave UV (366 nm) in the presence of ADP and Vi, kinase a ctivity was substantially decreased, and the MLCK polypeptide of 130 k Da was cleaved into several smaller fragments with apparent molecular masses of 100, 70, 60, 32, and 28 kDa, Inhibition of kinase activity a nd photocleavage were both competitively antagonized by the addition o f ATP, Inconsistency between the observed maximum levels of UV-induced inhibition of MLCK-mediated phosphorylation (80%) and photocleavage ( 15-20%) suggested that the photocleavage reaction proceeds as a two-st ep process, Monoclonal antibodies recognizing the C-terminus of MLCK l abeled the 60- and 28-kDa fragments, indicating that MLCK was cleaved at two sites, at 28 and 60 kDa from the C-terminus, within what are be lieved to be the autoinhibitory region and the catalytic site, respect ively. Moreover, Ca2+-calmodulin (Ca2+-CaM) protected against cleavage at the site at 28 kDa from the C-terminus, Analysis of the amino acid composition of the fragment revealed that the cleavage site at 28 kDa from C-terminus occurred at Lys 799 +/- 3 amino acid residues, which is in a region where the CaM-binding and pseudosubstrate regions overl ap. These results suggest that the three-dimensional structure of MLCK brings the regulatory segment into direct contact with the ATP-bindin g site, Moreover, the binding of Ca2+-CaM displaces the regulatory seg ment away from the catalytic site.