Isolation of cDNA for Bovine Stomach 155 kDa Protein Exhibiting Myosin Light Chain Kinase Activity1

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 ...

Section: Methodsmentioning

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

“…The role of myosin phosphorylation by MLCK in non-muscle cells is not well characterized but correlates with activities such as cell division, receptor capping, and platelet or endothelial cell contraction (Kerrick and Bourguignon, 1984;Kolodney and Wyslomerski, 1992;Wyslomerski and Lagunoff, 1990;Holzapfel et al, 1983; Ehrlich et al, 1991;Guiliano et al, 1992;Kolega and Taylor, 1993;Adelstein and Conti, 1975;Garcia et al, 1995). cDNAs representing vertebrate smooth and non-muscle MLCKs have been sequenced and the deduced amino acid sequences of the proteins compared Kobayashi et al, 1992;Shoemaker et al, 1990;Olson et al, 1990). This comparison reveals a high degree of sequence homology (>97% similarity) that extends beyond the central catalytic core and calmodulin-binding autoinhibitory region Kobayashi et al, 1992).…”

Section: Introductionmentioning

confidence: 99%

“…: 317-278-2146; Fax: 317-274-3318 regions outside of the catalytic core and autoinhibitory region are comprised of several class I and class II structural motifs that are similar to those in the related family of giant muscle proteins such as twitchin and titin (Olson et al, 1990;Labeit et al, 1992). The amino terminus of the mammalian MLCKs also contains a 175-residue insert not present in the avian smooth and non-muscle MLCKs Kobayashi et al, 1992). This insert is comprised of 15 tandem copies of a 12-residue repeat of unknown function.…”

mentioning

confidence: 99%

Expression of a Novel Myosin Light Chain Kinase in Embryonic Tissues and Cultured Cells

Gallagher

Garcia

Herring

1995

A novel, 208-kDa myosin light chain kinase (MLCK) distinct from smooth muscle and non-muscle MLCK has been identified by cross-reaction to two antibodies raised against smooth muscle MLCK. Additional antibodies directed against the amino and carboxyl termini of the smooth muscle MLCK do not react with the 208-kDa MLCK, suggesting these regions are distinct. 208-kDa MLCK phosphorylates 20-kDa myosin light chains in a Ca 2+ /calmodulin-dependent manner, consistent with it being a member of the MLCK family. Expression of 208-kDa MLCK and smooth muscle MLCK appears to be inversely regulated, with 208-kDa MLCK being most abundant during early development and declining at birth. In contrast, expression of smooth muscle MLCK is relatively low early during development and increases to become the predominant MLCK detected in all adult smooth and non-muscle tissues. The developmental expression pattern of the 208-kDa MLCK suggests this form be named, embryonic MLCK.Myosin light chain kinases (MLCK) 1 are Ca 2+ /calmodulin-regulated, actin and myosin binding, Ser/Thr protein kinases (Kamm and Stull, 1985;Sellers and Pato, 1984). In skeletal muscle, contraction is regulated by the troponin system and phosphorylation of regulatory light chain by skeletal muscle MLCK has a modulatory role in contraction-induced potentiation of isometric twitch tension (Sweeney et al., 1993). In smooth muscle, phosphorylation of the 20-kDa regulatory light chain of myosin by MLCK is a well characterized event, important for the initiation of contraction (Kamm and Stull, 1985). The role of myosin phosphorylation by MLCK in non-muscle cells is not well characterized but correlates with activities such as cell division, receptor capping, and platelet or endothelial cell contraction (Kerrick and Bourguignon, 1984;Kolodney and Wyslomerski, 1992;Wyslomerski and Lagunoff, 1990;Holzapfel et al., 1983; Ehrlich et al., 1991;Guiliano et al., 1992;Kolega and Taylor, 1993;Adelstein and Conti, 1975;Garcia et al., 1995). cDNAs representing vertebrate smooth and non-muscle MLCKs have been sequenced and the deduced amino acid sequences of the proteins compared Kobayashi et al., 1992;Shoemaker et al., 1990;Olson et al., 1990). This comparison reveals a high degree of sequence homology (>97% similarity) that extends beyond the central catalytic core and calmodulin-binding autoinhibitory region Kobayashi et al., 1992). The * This work was supported by American Cancer Society Grant IRG-161-H (to P. J. G.), a Grant-in-Aid from American Heart Association (to P. J. G. and B. P. H.), and a Grant-in-Aid from American Heart Association, Indiana Affiliate (to B. P. H.). ‡ To whom correspondence should be addressed: Dept. of Physiology and Biophysics, Indiana University School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5120. Tel.: 317-278-2146; Fax: 317-274-3318 regions outside of the catalytic core and autoinhibitory region are comprised of several class I and class II structural motifs that are similar to those in the related family of giant muscle p...

“…The extreme C-terminal kinase-related protein (KRP) domain, which binds myosin, is contained within both EC MLCK and SM MLCK but can be also expressed as an independent protein capable of stabilizing myofilaments in vitro (3,(21)(22)(23). The C-terminal half of the endothelial MLCK isoform (residues 923-1914) exhibits 99.8% homology to the human low molecular weight MLCK from hippocampus and substantial homology to published SM MLCK sequences from rabbit (94% homology), bovine (95% homology), and chicken (85% homology) (5,6,24,25). However, the exact biological function of the 922-amino acid N-terminal portion, which is unique to the high molecular weight MLCK isoform, is completely unknown.…”

mentioning

confidence: 99%

Differential Regulation of Alternatively Spliced Endothelial Cell Myosin Light Chain Kinase Isoforms by p60Src

Birukov

Csortos

Marzilli

et al. 2001

148

140

The Ca 2؉ /calmodulin-dependent endothelial cell myosin light chain kinase (MLCK) triggers actomyosin contraction essential for vascular barrier regulation and leukocyte diapedesis. Two high molecular weight MLCK splice variants, EC MLCK-1 and EC MLCK-2 (210 -214 kDa), in human endothelium are identical except for a deleted single exon in MLCK-2 encoding a 69-amino acid stretch ( 20 by Ca 2ϩ /calmodulin-dependent enzyme MLCK is essential for the initiation of nonmuscle and smooth muscle contraction (1, 3, 9 -11). In specific nonmuscle tissues, such as the vascular endothelium, this kinase is known to be involved in endothelial cell migration, cell retraction (12), endothelial cell barrier regulation (13), transendothelial migration of neutrophils (14, 15), and possibly apoptosis (16). The MLCK isoform abundantly expressed in smooth muscle (SM MLCK) generally exists as a 130-to 150-kDa protein that has been well characterized (for review see Refs. 3 and 11). However, Western blot screening of a variety of embryonic and adult smooth muscle and nonmuscle tissues revealed expression of a high molecular weight MLCK variant with electrophoretic mobility in the range of [208][209][210][211][212][213][214]. Garcia and colleagues (20) subsequently sequenced the high molecular weight MLCK isoform cloned from a human endothelial cell cDNA library, revealing an open reading frame, which encodes a protein of 1914 amino acids. Both the low molecular mass (130 -150 kDa) and high molecular mass (208 -214 kDa) MLCK isoforms share essentially identical actin binding, MLC binding, catalytic, and Ca 2ϩ /CaM-regulatory domains. The extreme C-terminal kinase-related protein (KRP) domain, which binds myosin, is contained within both EC MLCK and SM MLCK but can be also expressed as an independent protein capable of stabilizing myofilaments in vitro (3,(21)(22)(23). The C-terminal half of the endothelial MLCK isoform (residues 923-1914) exhibits 99.8% homology to the human low molecular weight MLCK from hippocampus and substantial homology to published SM MLCK sequences from rabbit (94% homology), bovine (95% homology), and chicken (85% homology) (5, 6, 24, 25). However, the exact biological function of the 922-amino acid N-terminal portion, which is unique to the high