Characterization of tightly associated smooth muscle myosin-myosin light-chain kinase-calmodulin complexes.
A current popular model to explain phosphorylation of smooth muscle myosin (SMM) by myosin light-chain kinase (MLCK) proposes that MLCK is bound tightly to actin but weakly to SMM. We found that MLCK and calmodulin (CaM) co-purify with unphosphorylated SMM from chicken gizzard, suggesting that they are tightly bound. Although the MLCK:SMM molar ratio in SMM preparations was well below stoichiometric (1:73+/-9), the ratio was approximately 23-37% of that in gizzard tissue. Fifteen to 30% of MLCK was associated with CaM at approximately 1 nM free [Ca(2+)]. There were two MLCK pools that bound unphosphorylated SMM with K(d) approximately 10 and 0.2 microM and phosphorylated SMM with K(d) approximately 20 and 0.2 microM. Using an in vitro motility assay to measure actin sliding velocities, we showed that the co-purifying MLCK-CaM was activated by Ca(2+) and phosphorylation of SMM occurred at a pCa(50) of 6.1 and at a Hill coefficient of 0.9. Similar properties were observed from reconstituted MLCK-CaM-SMM. Using motility assays, co-sedimentation assays, and on-coverslip enzyme-linked immunosorbent assays to quantify proteins on the motility assay coverslip, we provide strong evidence that most of the MLCK is bound directly to SMM through the telokin domain and some may also be bound to both SMM and to co-purifying actin through the N-terminal actin-binding domain. These results suggest that this MLCK may play a role in the initiation of contraction.
Hong
Feng
F
Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, 89557, USA.
Haldeman
Brian D
BD
John
Olivia A
OA
Brewer
Paul D
PD
Wu
Yi-Ying
YY
Ni
Shaowei
S
Wilson
David P
DP
Walsh
Michael P
MP
Baker
Jonathan E
JE
Cremo
Christine R
CR
D000199:Actins / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002147:Calmodulin / Q000378:metabolism*; D002417:Cattle; D002645:Chickens; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000378:metabolism*; D018994:Myosin Light Chains / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D010446:Peptide Fragments / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D024745:Smooth Muscle Myosins / Q000378:metabolism*
D000199:Actins; D002147:Calmodulin; D046912:Multiprotein Complexes; D018994:Myosin Light Chains; D010446:Peptide Fragments; C060640:telokin; D000255:Adenosine Triphosphate; D009219:Myosin-Light-Chain Kinase; D024745:Smooth Muscle Myosins; D008274:Magnesium