Abstract
The role of the NH2-terminal domain of the 20,000-dalton light chain on the regulatory function of smooth muscle myosin was studied by exchanging it in myosin with various mutant forms. The 10 S to 6 S conformational transition as well as the thick filament formation were significantly influenced by the deletion or substitution of the amino acid residues at the NH2-terminal side of the phosphorylation site (Ser19). Whereas the deletion of Ser1-Thr10 did not significantly affect these functions, further deletion of Lys11-Arg16 completely abolished the formation of 10 S conformation and induced thick filament formation. Among the residues in this region, Arg13 and Arg16 were most important for these functions since substitution of these residues by Glu or Ala significantly altered these functions. Similar substitutions of Lys11 and Lys12 also stabilized the 6 S conformation and thick filament formation but less effectively. While the 6 S conformation was stabilized, the deletion of NH2-terminal residues did not activate the actin-activated ATPase activity. This suggests that stabilization of the 6 S conformation is not directly coupled with activation of actomyosin ATPase activity but rather a more defined conformational change around the phosphorylation site is necessary for activation. Such a change also influences the 6 S to 10 S conformation and, therefore, the filament formation. To support this notion, substitution of Lys11 and Lys12 by Glu-Glu inhibited the phosphorylation-induced activation of actomyosin ATPase activity.
Highlights
Were significantly influenced by the deletion or substi- makes it unlikely that its phosphorylation site can interact tution of the amino acid residues at the NH,terminal directly with the ATPase or actin binding sites since both of side of the phosphorylation site (SerlS)
The contractile apparatus of vertebrate smooth muscle cells 10 S myosin and an extended formknown as 6 S myosin as well as nonmuscle cells is regulated by phosphorylation of (Trybus et al, 1982; Onishi and Wakabayashi, 1982; Craig et the regulatory light chain of myosin, whichcauses activation of al., 1983; Suzuki et al, 1978).The former has a very low level actomyosinATPase activity (Hartshorne, 1987; Sellers and of ATPase activity in thepresence of Ca2+and Mg2' (Ikebeet al., Adelstein, 1987; Kammand Stull, 1989)by a Ca2+/calmodulin- 1983) since the nucleotide becomes noncovalently trapped in dependent protein kinase, myosin light chain kinase (MLC the protein (Cross et al, 1986)
Expression of Recombinant Mutant Proteins of the Myosin Regulatory Light Chain-The recombinant light chain cloned into theexpression vector pT7-7 was expressed in Escherichia coli strain BL21 (DE3).The NH,terminal tag derived from the vector sequence, i.e. 6 amino acid residues at theNH,terminal end of the recombinantlightchain,was removedfrom the regulatory light chain expression vector because the solubility of the purified light chain with the NH,terminal tag was often low, which resulted in a lower final yield
Summary
Materials-Smooth muscle myosin was prepared from frozen turkey gizzards as described previously (Ikebe and Hartshorne, 1985~)A. ctin was prepared from rabbit skeletal muscle acetone powder according to Spudich and Watt (1971).Other materialswere obtained a s described in the accompanying paper (Ikebe et ul., 1994). The myosin exchanged with the recombinant light chains was dialyzed against 30 mM Tris-HC1(pH 7.5), mM MgCl,, and 1mM DTT a t 4 “C for 3 h. Myosin containing various mutant light chains in 0.4 M KC1 was diluted with buffer containing 10 mM MgCl,, 2 mM Dl”, 0.5 mM ATP, and 10 mM Tris-HC1(pH 7.5) to 60 mM KC1 (final) and centrifuged by an Airfuge (Beckman) a t 100,000 x g for 20 min, and the myosin concentration of supernatant was estimated by K+ EDTA ATPase activity a s described previously (Higashiharaet al., 1989).Filament formation was confirmed by SDS-PAGE of supernatant and pellet followed by densitometric scanning of myosin heavy chain.
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