Autophosphorylation of Smooth Muscle Myosin Light Chain Kinase at Its Regulatory Domain

Tokui, Toshiya; Ando, Shoji; Ikebe, Mitsuo

doi:10.1021/bi00015a031

Cited by 34 publications

(37 citation statements)

References 43 publications

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“…The 32 P incorporation into MLCK-1 was essentially abolished by the specific p60 src kinase inhibitor PP-2 (250 nM). However, all three MLCK isoforms exhibited low level of 32 P incorporation even in the absence of p60 src (0.63 Ϯ 0.21 mol of PO 4 /mol of protein) consistent with the MLCK autophosphorylation previously described for smooth muscle MLCK (39,40). This was confirmed by heat treatment of the MLCK preparations (70°C for 5 min), which completely abolished incorporation of 32 P into MLCK in the absence of p60 src (data not shown).…”

Section: In Vitro Phosphorylation Of Mlck Isoforms By P60supporting

confidence: 83%

“…Finally, mass spectrometry analysis of the fragments obtained after cyanogen bromide cleavage of the MLCK-1 suggested that the two phosphate groups are contained within the Lys 1721 -Met 1761 EC MLCK fragment (data not shown) consistent with the presence of the EC MLCK-1 autophosphorylation sites Thr 1748 and Ser 1760 , which are homologous to the previously described SM MLCK autophosphorylation sites Thr 803 and Ser 815 (39).…”

Section: Identification Of P60supporting

confidence: 66%

“…In turn, phosphorylation by p21-activated kinase decreases MLCK-1 catalytic activity by ϳ50% via decrease in maximum velocity (V max ) without affecting K CaM (47). In addition, Thr 803 , Ser 815 , and Ser 823 of the smooth muscle MLCK isoform undergo autophosphorylation in vitro also resulting in decreased MLCK affinity to Ca 2ϩ /calmodulin (39).…”

Section: Discussionmentioning

confidence: 99%

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

Birukov

Csortos

Marzilli

et al. 2001

Journal of Biological Chemistry

147

140

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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

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Section: In Vitro Phosphorylation Of Mlck Isoforms By P60supporting

confidence: 83%

Section: Identification Of P60supporting

confidence: 66%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

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

Birukov

Csortos

Marzilli

et al. 2001

Journal of Biological Chemistry

147

140

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“…It is known that calmodulin-dependent protein kinase II is significantly activated by autophosphorylation at Thr 287 , which lies near the autoinhibitory region of the kinase (53)(54)(55). The autophosphorylation-induced activation of the protein kinase activity is also known for myosin light chain kinase, in which the autophosphorylation site is found to be in close proximity to the autoinhibitory region (56). Because autophosphorylation is the intramolecular process, the rate of autophosphorylation is independent to myosin III concentration, and therefore the observed rate in the present study probably represents the rate in the cell environment.…”

Section: Discussionmentioning

confidence: 99%

Determination of Human Myosin III as a Motor Protein Having a Protein Kinase Activity

Komaba

Inoue

Maruta

et al. 2003

Journal of Biological Chemistry

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The class III myosin is the most divergent member of the myosin superfamily, having a domain with homology to a protein kinase. However, the function of class III myosin at a molecular level is not known at all, and it has been questioned whether it is actually an actinbased motor molecule. Here, we showed that human myosin III has an ATPase activity that is significantly activated by actin (20-fold) with K actin of 112 M and V max of 0.34 s ؊1 , indicating the mechanoenzymatic activity of myosin III. Furthermore, we found that human myosin III has actin translocating activity (0.11 ؎ 0.05 m/s) using an in vitro actin gliding assay, and it moves toward the plus end of actin filaments. Myosin III containing calmodulin as the light chain subunit showed a protein kinase activity and underwent autophosphorylation. The autophosphorylation was the intramolecular process, and the sites were at the C-terminal end of the motor domain. Autophosphorylation significantly activated the kinase activity, although it did not affect the ATPase activity. The present study is the first report that clearly demonstrates that the class III myosin is an actin-based motor protein having a protein kinase activity.Myosin III is a member of the myosin superfamily, which consists of at least 18 classes (1-5). The class III myosin represents the most divergent member of the myosin superfamily. The motor domain of myosin III shows ϳ24 -27% sequence identity to myosin Is and ϳ22-26% sequence identity to myosin-IIs (6). Of particular interest is that myosin III has an amino terminus domain that resembles to protein kinases. This domain contains the characteristic motifs for protein kinases such as a glycine-rich loop, an invariable Lys residue required for ATP binding, and a catalytic loop. Given its high degree of divergence, if myosin III evolved at the same rate as other myosins, the myosin III lineage would predate the divergence of yeast. Class III myosin was originally found in Drosophila photoreceptor cells and subsequently found in vertebrates including human myosin III (7,8).The function of Myosin III is best studied in Drosophila photoreceptor cells. Each photoreceptor cell has a specialized organelle consisting of a stack of microvilli known as a rhabdomere. The phototransduction machinery is localized in the rhabdomere (9). Drosophila photoreceptors undergo a prolonged depolarization afterpotential that persists after cessation of the light stimulus. Prolonged depolarization afterpotential results from the stable conversion of rhodopsin to the light-activated form, metarhodopsin, in response to blue light. During a prolonged depolarization afterpotential, photoreceptor cells become refractory to subsequent prolonged depolarization afterpotential-inducing stimuli and are inactivated. Mutants that are defective for both inactivation and the prolonged depolarization afterpotential are known as neither inactivation nor afterpotential (nina) mutants (10, 11), and the myosin III was identified (12) as one of eight nina complementation gr...

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“…Wehave shown that mitotic Hela cell extracts have higher smMLCK kinase activities than nonmitotic cell extracts (1 1). This mitosis-specific kinase activity is thought to be cdc2 ki- Furthermore, autophosphorylation of smMLCK has been reported (6,7,23 pmol) in a final volume of 140 fA. Twenty fA of aliquots were withdrawn at the indicated times and mixed with 5 x SDSsample buffer to stop the reactions.…”

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confidence: 99%

Activation of Chicken Gizzard Myosin Light Chain Kinase by Ca2+/Calmodulin is Inhibited by Autophosphorylation.

Abe

Hasegawa

Hosoya

1996

Cell Struct. Funct.

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Keywords: smooth muscle myosin light chain kinase/autophosphorylation/chicken gizzard/MLCK ABSTRACT.Myosin light chain kinase (MLCK)is a calmodulin-dependent protein kinase which phosphorylates the 20,000 dalton regulatory light chain of myosin II. Here we show that activation of chicken gizzard MLCKby Ca2+/calmodulin is inhibited by autophosphorylation at 2 sites in the absence of Ca2+/calmodulin. Two phosphorylation sites are located in the functional domain of the kinase, the threonine site toward the actin binding domain near the N-terminus of MLCKand the serine site in immediate proximity to the calmodulin binding site. The autophosphorylation was significantly inhibited by the binding of calmodulin to MLCKin the presence of Ca2+.

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Autophosphorylation of Smooth Muscle Myosin Light Chain Kinase at Its Regulatory Domain

Cited by 34 publications

References 43 publications

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

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

Determination of Human Myosin III as a Motor Protein Having a Protein Kinase Activity

Activation of Chicken Gizzard Myosin Light Chain Kinase by Ca2+/Calmodulin is Inhibited by Autophosphorylation.

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