Myosin light chain kinase stimulates smooth muscle myosin ATPase activity by binding to the myosin heads without phosphorylating the myosin light chain.
Myosin light chain kinase (MLCK) is a multifunctional regulatory protein of smooth muscle contraction [IUBMB Life 51 (2001) 337, for review]. The well-established mode for its regulation is to phosphorylate the 20 kDa myosin light chain (MLC 20) to activate myosin ATPase activity. MLCK exhibits myosin-binding activity in addition to this kinase activity. The myosin-binding activity also stimulates myosin ATPase activity without phosphorylating MLC 20 [Proc. Natl. Acad. Sci. USA 96 (1999) 6666]. We engineered an MLCK fragment containing the myosin-binding domain but devoid of a catalytic domain to explore how myosin is stimulated by this non-kinase pathway. The recombinant fragment thus obtained stimulated myosin ATPase activity by V(max)=5.53+/-0.63-fold with K(m)=4.22+/-0.58 microM (n=4). Similar stimulation figures were obtained by measuring the ATPase activity of HMM and S1. Binding of the fragment to both HMM and S1 was also verified, indicating that the fragment exerts stimulation through the myosin heads. Since S1 is in an active form regardless of the phosphorylated state of MLC 20, we conclude that the non-kinase stimulation is independent of the phosphorylating mode for activation of myosin.
Gao
Ying
Y
Department of Pharmacology, Gunma University School of Medicine, Maebashi, Gunma 371-8511, Japan.
Kawano
Kazufumi
K
Yoshiyama
Shinji
S
Kawamichi
Hozumi
H
Wang
Xiaoming
X
Nakamura
Akio
A
Kohama
Kazuhiro
K
D001665:Binding Sites; D004789:Enzyme Activation; D005982:Glutathione Transferase / Q000235:genetics; D018994:Myosin Light Chains / Q000378:metabolism; D015879:Myosin Subfragments; D009219:Myosin-Light-Chain Kinase / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D024745:Smooth Muscle Myosins / Q000737:chemistry* / Q000378:metabolism*
D018994:Myosin Light Chains; D015879:Myosin Subfragments; D011993:Recombinant Fusion Proteins; D005982:Glutathione Transferase; D009219:Myosin-Light-Chain Kinase; D024745:Smooth Muscle Myosins