Kinetic and motor functions mediated by distinct regions of the regulatory light chain of smooth muscle myosin.
To understand the importance of selected regions of the regulatory light chain (RLC) for phosphorylation-dependent regulation of smooth muscle myosin (SMM), we expressed three heavy meromyosins (HMMs) containing the following RLC mutants; K12E in a critical region of the phosphorylation domain, GTDP(95-98)/AAAA in the central hinge, and R160C a putative binding residue for phosphorylated S19. Single-turnover actin-activated Mg(2+)-ATPase (V(max) and K(ATPase)) and in vitro actin-sliding velocities were examined for both unphosphorylated (up-) and phosphorylated (p-) states. Turnover rates for the up-state (0.007-0.030 s(-1)) and velocities (no motion) for all constructs were not significantly different from the up-wild type (WT) indicating that they were completely turned off. The apparent binding constants for actin in the presence of ATP (K(ATPase)) were too weak to measure as expected for fully regulated constructs. For p-HMM containing GTDP/AAAA, we found that both ATPase and motility were normal. The data suggest that the native sequence in the central hinge between the two lobes of the RLC is not required for turning the HMM off and on both kinetically and mechanically. For p-HMM containing R160C, all parameters were normal, suggesting that R160C is not involved in coordination of the phosphorylated S19. For p-HMM containing K12E, the V(max) was 64% and the actin-sliding velocity was approximately 50% of WT, suggesting that K12 is an important residue for the ability to sense or to promote the conformational changes required for kinetic and mechanical activation.
Ni
Shaowei
S
Department of Biochemistry and Molecular Biology, University of Nevada School of Medicine, Reno, NV 89557, USA.
Hong
Feng
F
Brewer
Paul D
PD
Ikebe
Mitsuo
M
Onishi
Hirofumi
H
Baker
Jonathan E
JE
Facemyer
Kevin C
KC
Cremo
Christine R
CR
D019943:Amino Acid Substitution; D000818:Animals; D007700:Kinetics; D020409:Molecular Motor Proteins / Q000235:genetics; D018994:Myosin Light Chains / Q000235:genetics / Q000502:physiology*; D015879:Myosin Subfragments / Q000235:genetics / Q000502:physiology; D010766:Phosphorylation; D017434:Protein Structure, Tertiary; D024745:Smooth Muscle Myosins / Q000235:genetics / Q000502:physiology*
D020409:Molecular Motor Proteins; D018994:Myosin Light Chains; D015879:Myosin Subfragments; D024745:Smooth Muscle Myosins