Stimulation of the ATP‐dependent interaction between actin and myosin by a myosin‐binding fragment of smooth muscle caldesmon

Lin, Yuan; Ishikawa, Ryoki; Okagaki, Tsuyoshi; Ye, Lihong; Kohama, Kazuhiro

doi:10.1002/cm.970290308

Cited by 23 publications

(15 citation statements)

References 48 publications

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“…We ensured that the rate of ATP hydrolysis was steady and that the specific activity of the ATPase did not differ from our previously reported values. Thus, we were able to express the activity as a normalized ATPase activity (21). V max and K m were obtained by double reciprocal plots.…”

Section: Methodsmentioning

confidence: 99%

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

Kishi

Nakamura

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Methodsmentioning

confidence: 99%

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

Kishi

Nakamura

et al. 1999

Proc. Natl. Acad. Sci. U.S.A.

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“…We ensured that the rate of ATP hydrolysis was constant and that the specific activity of the ATPase did not differ from our previously reported values. Therefore, we expressed the activity as a normalized ATPase activity (27).…”

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confidence: 99%

The Structure and Function of the Actin-binding Domain of Myosin Light Chain Kinase of Smooth Muscle

Ye¹,

Hayakawa²,

Kishi³

et al. 1997

Journal of Biological Chemistry

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In addition to its kinase activity, the myosin light chain kinase (MLCK) of smooth muscle has an actin binding activity through which it can regulate the actinmyosin interaction of smooth muscle (Kohama, K., Okagaki, T., Hayakawa, K., Lin, Y., Ishikawa, R., Shimmen In addition to this kinase activity, MLCK can act as an actin-binding protein; MLCK is present in association with the sarcomeric I-band (2), and it binds to actin filaments with a high affinity (3-5). We have shown that MLCK can inhibit the ATP-dependent interaction between actin and myosin by binding to actin filaments. This inhibition can be relieved by Ca 2ϩ /CaM, as was demonstrated by avoiding the complication derived from its kinase activity (6 -8), although the demonstration was limited to in vitro only.Such an inhibition, however, is not peculiar to MLCK. Caldesmon (see Ref. 9 for a review) and calponin (10) The relationship between the structure of MLCK and the function of the kinase activity in phosphorylating the myosin light chain and how Ca 2ϩ /CaM modulates the activity has been well established (see Ref. 13 for a review). However, there has been little study of the structure-function relationship of the actin binding activity except for the purification of an actinbinding fragment from MLCK (14).In this study, we have investigated which sequence of MLCK is responsible for the actin binding activity, which sequence inhibits the actin-myosin interaction, and which sequence binds CaM to relieve the inhibition. Our approach has been to cleave MLCK to prepare native fragments containing the actin binding activity (14), to design MLCK cDNA to express the recombinant fragments of actin binding activity in Escherichia coli, and then to analyze them biochemically. A few peptides have been synthesized to confirm these analyses. We have shown that: (i) MLCK has two actin-binding sites on the Nterminal side away from the central kinase domain, (ii) the binding site responsible for the inhibitory effect is at the Met MATERIALS AND METHODSPreparation of Proteins-All procedures were carried out at 0 -4°C. The purity of proteins was routinely monitored by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (see below) so * This work was supported in part by grants from the Fujisawa Foundation and the Mitsubishi Foundation and by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.ʈ To whom correspondence should be addressed. Tel.: 81-27-220-7960; Fax: 81-27-235-1401 or 81-27-220-7962. 1 The abbreviations used are: MLCK, myosin light chain kinase; CaM, calmodulin; PAGE, polyacrylamide gel electrophoresis; NTCB fragment, chicken gizzard MLCK fragment produced by the cleavage with 2-nitro-5-thiocyanatobenzoic acid; NN fragment, expressed fragment (amino acids 1-337 ...

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“…We have reported that caldesmon (29) and calponin (30) exert a stimulatory effect on the myosin ATPase activity of smooth muscle through myosinbinding activity. Similarly, we recently observed that the myosinbinding fragment of MLCK, which is devoid of kinase activity, stimulated the myosin ATPase activity (13).…”

Section: Discussionmentioning

confidence: 99%

Role of the short isoform of myosin light chain kinase in the contraction of cultured smooth muscle cells as examined by its down-regulation

Bao¹,

Oishi²,

Yamada³

et al. 2002

Proc. Natl. Acad. Sci. U.S.A.

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Stimulation of the ATP‐dependent interaction between actin and myosin by a myosin‐binding fragment of smooth muscle caldesmon

Cited by 23 publications

References 48 publications

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

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

The Structure and Function of the Actin-binding Domain of Myosin Light Chain Kinase of Smooth Muscle

Role of the short isoform of myosin light chain kinase in the contraction of cultured smooth muscle cells as examined by its down-regulation

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