title: Myosin light chain kinase steady-state kinetics: comparison of smooth muscle myosin II and nonmuscle myosin IIB as substrates.
abstract: Myosin light chain kinase (MLCK) phosphorylates S19 of the myosin regulatory light chain (RLC), which is required to activate myosin's ATPase activity and contraction. Smooth muscles are known to display plasticity in response to factors such as inflammation, developmental stage, or stress, which lead to differential expression of nonmuscle and smooth muscle isoforms. Here, we compare steady-state kinetics parameters for phosphorylation of different MLCK substrates: (1) nonmuscle RLC, (2) smooth muscle RLC, and heavy meromyosin subfragments of (3) nonmuscle myosin IIB, and (4) smooth muscle myosin II. We show that MLCK has a ~2-fold higher kcatfor both smooth muscle myosin II substrates compared with nonmuscle myosin IIB substrates, whereas Kmvalues were very similar. Myosin light chain kinase has a 1.6-fold and 1.5-fold higher specificity (kcat/Km) for smooth versus nonmuscle-free RLC and heavy meromyosin, respectively, suggesting that differences in specificity are dictated by RLC sequences. Of the 10 non-identical RLC residues, we ruled out 7 as possible underlying causes of different MLCK kinetics. The remaining 3 residues were found to be surface exposed in the N-terminal half of the RLC, consistent with their importance in substrate recognition. These data are consistent with prior deletion/chimera studies and significantly add to understanding of MLCK myosin interactions.
  
  
  CONCLUSIONS
  Phosphorylation of nonmuscle and smooth muscle myosin by myosin light chain kinase (MLCK) is required for activation of myosin's ATPase activity. In smooth muscles, nonmuscle myosin coexists with smooth muscle myosin, but the two myosins have very different chemo-mechanical properties relating to their ability to maintain force. Differences in specificity of MLCK for different myosin isoforms had not been previously investigated. We show that the MLCK prefers smooth muscle myosin by a significant factor. These data suggest that nonmuscle myosin is phosphorylated more slowly than smooth muscle myosin during a contraction cycle.
authors.affiliation: Department of Pharmacology, University of Nevada Reno School of Medicine, Reno, Nevada, USA.
authors.forename: Diego B
authors.initials: DB
authors.lastname: Alcala
authors.affiliation: Department of Pharmacology, University of Nevada Reno School of Medicine, Reno, Nevada, USA.
authors.forename: Brian D
authors.initials: BD
authors.lastname: Haldeman
authors.affiliation: Department of Pharmacology, University of Nevada Reno School of Medicine, Reno, Nevada, USA.
authors.forename: Richard K
authors.initials: RK
authors.lastname: Brizendine
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
authors.forename: Agata K
authors.initials: AK
authors.lastname: Krenc
authors.affiliation: Department of Pharmacology, University of Nevada Reno School of Medicine, Reno, Nevada, USA.
authors.forename: Josh E
authors.initials: JE
authors.lastname: Baker
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Chicago, Chicago, Illinois, USA.
authors.forename: Ronald S
authors.initials: RS
authors.lastname: Rock
authors.affiliation: Department of Pharmacology, University of Nevada Reno School of Medicine, Reno, Nevada, USA. cremo@unr.edu.
authors.forename: Christine R
authors.identifier: http://orcid.org/0000-0001-5516-5222
authors.initials: CR
authors.lastname: Cremo
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D007700:Kinetics; D008958:Models, Molecular; D015879:Myosin Subfragments / Q000737:chemistry / Q000378:metabolism; D009219:Myosin-Light-Chain Kinase / Q000737:chemistry / Q000378:metabolism*; D024501:Nonmuscle Myosin Type IIB / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D024745:Smooth Muscle Myosins / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D015879:Myosin Subfragments; D009219:Myosin-Light-Chain Kinase; D024501:Nonmuscle Myosin Type IIB; D024745:Smooth Muscle Myosins
keywords: myosin light chain kinase; myosin regulatory light chain; nonmuscle myosin; phosphorylation; smooth muscle; smooth muscle myosin; steady-state kinetics