Myosin light-chain kinase of smooth muscle stimulates myosin ATPase activity without phosphorylating myosin light chain

Abstract

Myosin light-chain kinase (MLCK) of smooth muscle is multifunctional, being composed of N-terminal actinbinding domain, central kinase domain, and C-terminal myosinbinding domain. The kinase domain is the best characterized; this domain activates the interaction of smooth-muscle myosin with actin by phosphorylating the myosin light chain. We have recently shown that the Met-1-Pro-41 sequence of MLCK binds to actin to inhibit this interaction. However, it is not known whether the myosin-binding domain modifies the actin-myosin interaction. We designed MLCK⅐cDNA to overexpress the Asp-777-Glu-972 sequence in Escherichia coli. The purified Asp-777-Glu-972 fragment, although devoid of the kinase activity, exerted a stimulatory effect on the ATPase activity of dephosphorylated myosin (V max ‫؍‬ 7.36 ؎ 0.44-fold, K m ‫؍‬ 1.06 ؎ 0.20 M, n ‫؍‬ 4). When the N-terminal 39 residues of the fragment were deleted from the fragment, the resultant fragment, Met-816-Glu-972, lost the stimulatory activity. We synthesized the Ala-777-Ser-815 peptide that was deleted from the fragment and confirmed its stimulatory effect of the peptide (V max ‫؍‬ 3.03 ؎ 0.22-fold, K m ‫؍‬ 6.93 ؎ 1.61 M, n ‫؍‬ 3). When this peptide was further divided into Asp-777-Met-795 and Ala-796-Ser-815 peptides, the stimulatory activity was found in the latter. We confirmed that the myosin phosphorylation did not occur during the experiments with the above fragments and peptides. Therefore, we suggest that phosphorylation is not obligatory for smooth-muscle myosin not to be active.Myosin light-chain kinase (MLCK) is an enzyme that phosphorylates the light chain of myosin in the presence of Ca 2ϩ and calmodulin (Ca͞CaM) (see refs. 1 and 2 for reviews). This catalytic domain is in the central part of MLCK neighbored by the regulatory domain that binds Ca͞CaM. The N-terminal portion consists of actin-binding domains. At the Met-1-Pro-41 sequence, MLCK binds actin filaments (3) in a Ca͞CaM-dependent manner (4). The Ca͞CaM-independent actin-binding site is located somewhere between the Ca͞CaM-dependent site and the catalytic domain (4). These actin-binding sites are expected to bundle actin filaments (5). The C terminus of MLCK is present in smoothmuscle cells as a gene product called telokin, which is independent of MLCK expression (6). Because telokin binds myosin to assemble it into thick filaments (7), the C terminus of MLCK is considered to be a myosin-binding domain.The myosin phosphorylated by the catalytic domain of MLCK is in an active form and interacts with actin filaments to contract smooth muscle. This mode of phosphorylation is widely accepted as the regulatory path for actin-myosin interaction (see refs. 1 and 2 for reviews). However, there are a number of observations that are not explained by the mode of phosphorylation (see ref. 8 for review). To solve this problem, Ebashi and colleagues have proposed that MLCK can activate the actin-myosin interaction without phosphorylating myosin (9-11).To search for the domain that is responsible for t...

LFA-1-induced T cell migration on ICAM-1 involves regulation of MLCK-mediated attachment and ROCK-dependent detachment

“…The only recognized substrate of MLCK is MLC, which is phosphorylated on Thr18 Ser19, thereby activating myosin ATPase activity (reviewed in Bresnick, 1999). There is also evidence in smooth muscle cells that the actin-bundling activity of MLCK, rather than the kinase activity, is dominant in membrane ruffling (Kishi et al, 2000;Ye et al, 1999). The inhibitor ML-7 and the MLCK autoinhibitory peptide used in this study both target the kinase domain and block catalytic activity of MLCK (Saitoh et al, 1987;Tanaka et al, 1995).…”

Stimulatory effects of arachidonic acid on myosin ATPase activity and contraction of smooth muscle via myosin motor domain

“…Mant-ATP at 0.25 M was mixed with 0.5 M myosin in the unphosphorylated form for mant-ATP fluorescence measurements in buffer consisting of 60 mM KCl, 5 mM MgCl 2, 0.2 mM EGTA, 1 mM DTT, and 20 mM Tris ⅐ HCl (pH 7.5) in the vehicle or various concentrations of AA (61). Changes in the fluorescence due to the binding to and release from the myosin were detected as the energy transfer from tryptophan excited at 290 nm, and the emitted light at 440 nm was recorded by a fluorometer (F-4500; Hitachi, Tokyo, Japan).…”

Is myosin light-chain phosphorylation a regulatory signal for the osmotic activation of the Na+-K+-2Cl− cotransporter?

“…MLCK has been shown to alter ion transport through both MLC phosphorylation-dependent and -independent mechanisms (50). Furthermore, whether MLC phosphorylation or merely myosin activity is involved in transporter regulation remains questionable, because the latter can be regulated in a phosphorylation-independent manner as well (14,54). To approach these issues, we targeted MLC phosphorylation per se rather than MLCK activity.…”

“…Even if such a relationship exists, however, it remains to be determined whether an increase in myosin phosphorylation is in fact a signal for the osmotic activation of NKCC or whether a basal level of MLC phosphorylation (or myosin ATPase activity) is merely a permissive factor necessary for the maximal osmotic stimulation of the cotransporter. Finally, MLCK-mediated regulation does not necessarily occur through myosin phosphorylation, because MLCK can activate myosin ATPase activity without phosphorylation (14,54), and MLCK has been reported to act on transporters in a myosin-independent manner (50).…”