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Query: UNIPROT:P06889 (Mol)
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It has been established for many years that MLCK is regulated by the intracellular Ca2+ concentration via the formation of the Ca2+ -calmodulin-MLCK complex. A more recent discovery has been that the myosin phosphatase may also be regulated. This is manifest at suboptimal Ca2+ levels where under certain conditions (e.g. stimulation with several agonists) the MP is inhibited. The net result being that the extent of myosin phosphorylation for a fixed Ca2+ level is increased, i.e. an enhanced Ca2+ -sensitivity. Spurred by this intriguing discovery several laboratories began studies on MP with an emphasis to determine the regulatory, or inhibitory, mechanism. A similar preparation was obtained by 3 laboratories and consisted of a catalytic subunit, PP1delta, plus a large subunit (M130/133 for gizzard, M130 for bladder and M110 for rat aorta) and a smaller subunit of 20-21 kD. The isolated catalytic subunit has a much lower activity towards phosphorylated myosin than the holoenzyme, thus the non-catalytic subunits may serve as targeting proteins and in addition may play a regulatory role. Because of the difference in activities between the catalytic subunit and holoenzyme, one mechanism of regulation may involve dissociation of the trimeric complex, and such was proposed for the effect of arachidonic acid. Another suggested regulatory mechanism was that phosphorylation of the large subunit in its C-terminal half caused inhibition of phosphatase activity. The two mechanisms need not be mutually exclusive and in addition several kinases could influence the activity of the myosin phosphatase. In order to understand the molecular basis of phosphatase regulation it is necessary to determine the topography of the holoenzyme and identify sites of interaction between subunits and substrate. This work is in progress. Using various truncation mutants of M130/133 it has been determined that the binding sites for both PP1c and substrate are located within the N-terminal part of the molecule. The M20 subunit binds to the C-terminal end, although the functional significance of this is not established. Many questions remain to be answered concerning the biochemistry of the myosin phosphatase. An exciting and challenging focus will be to determine the mechanism(s) of regulation and to unravel the signaling cascade(s) that are initiated by agonist-receptor complex formation. In addition, the location of the MP is not known and it is important to establish which (if any) of the cytoskeletal elements are involved in binding to MP. Finally, it is assumed that the trimeric phosphatase, as discussed above, is specific for myosin dephosphorylation and does not act on other substrates. Because of the breadth of its distribution in different tissues and the wide range of proteins interacting with the ankyrin repeats it is possible that this phosphatase, or variants thereof, has roles in other cellular processes.
Mol Cell Biochem 1999 Jan
PMID:Interactions of protein phosphatase type 1, with a focus on myosin phosphatase. 1009 73

The myosin light chain kinase (MLCK) gene, a muscle member of the immunoglobulin gene superfamily, yields both smooth muscle and nonmuscle cell isoforms. Both isoforms are known to regulate contractile activity via calcium/calmodulin-dependent myosin light chain phosphorylation. We previously cloned from a human endothelial cell (EC) cDNA library a high-molecular-weight nonmuscle MLCK isoform (EC MLCK (MLCK 1) with an open reading frame that encodes a protein of 1914 amino acids. We now describe four novel nonmuscle MLCK isoforms (MLCK 2, 3a, 3b, and 4) that are the alternatively spliced variants of an mRNA precursor that is transcribed from a single human MLCK gene. The primary structure of the cDNA encoding the nonmuscle MLCK isoform 2 is identical to the previously published human nonmuscle MLCK (MLCK 1) (J. G. N. Garcia et al., 1997, Am. J. Respir. Cell Mol. Biol. 16, 489-494) except for a deletion of nucleotides 1428-1634 (D2). The full nucleotide sequence of MLCK isoforms 3a and 3b and partial sequence for MLCK isoform 4 revealed identity to MLCK 1 except for deletions at nucleotides 5081-5233 (MLCK 3a, D3), double deletions of nucleotides 1428-1634 and 5081-5233 (MLCK 3b), and nucleotide deletions 4534-4737 (MLCK 4, D4). Northern blot analysis demonstrated the extended expression pattern of the nonmuscle MLCK isoform(s) in both human adult and human fetal tissues. RT-PCR using primer pairs that were designed to detect specifically nonmuscle MLCK isoforms 2, 3, and 4 deletions (D2, D3, and D4) confirmed expression in both human adult and human fetal tissues (lung, liver, brain, and kidney) and in human endothelial cells (umbilical vein and dermal). Furthermore, relative quantitative expression studies demonstrated that the nonmuscle MLCK isoform 2 is the dominant splice variant expressed in human tissues and cells. Further analysis of the human MLCK gene revealed that the MLCK 2 isoform represents the deletion of an independent exon flanked by 5' and 3' neighboring introns of 0.6 and 7.0 kb, respectively. Together these studies demonstrate for the first time that the human MLCK gene yields multiple nonmuscle MLCK isoforms by alternative splicing of its transcribed mRNA precursor with differential distribution of these isoforms in various human tissues and cells.
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PMID:A single human myosin light chain kinase gene (MLCK; MYLK). 1019 65

A novel myosin light chain kinase (MLCK) cDNA was isolated from a HeLa cell cDNA library. The deduced amino acid sequence was identical to that of a zipper-interacting protein kinase (ZIPK) which mediates apoptosis [Kawai et al. (1998) Mol. Cell. Biol. 18, 1642-1651]. Here we found that HeLa ZIPK phosphorylated the regulatory light chain of myosin II (MRLC) at both serine 19 and threonine 18 in a Ca2+/calmodulin independent manner. Phosphorylation of myosin II by HeLa ZIPK resulted in activation of actin-activated MgATPase activity of myosin II. HeLa ZIPK is the first non-muscle MLCK that phosphorylates MRLC at two sites.
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PMID:ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells. 1035 87

Sea urchin coelomocytes represent an excellent experimental model system for studying retrograde flow. Their extreme flatness allows for excellent microscopic visualization. Their discoid shape provides a radially symmetric geometry, which simplifies analysis of the flow pattern. Finally, the nonmotile nature of the cells allows for the retrograde flow to be analyzed in the absence of cell translocation. In this study we have begun an analysis of the retrograde flow mechanism by characterizing its kinetic and structural properties. The supramolecular organization of actin and myosin II was investigated using light and electron microscopic methods. Light microscopic immunolocalization was performed with anti-actin and anti-sea urchin egg myosin II antibodies, whereas transmission electron microscopy was performed on platinum replicas of critical point-dried and rotary-shadowed cytoskeletons. Coelomocytes contain a dense cortical actin network, which feeds into an extensive array of radial bundles in the interior. These actin bundles terminate in a perinuclear region, which contains a ring of myosin II bipolar minifilaments. Retrograde flow was arrested either by interfering with actin polymerization or by inhibiting myosin II function, but the pathway by which the flow was blocked was different for the two kinds of inhibitory treatments. Inhibition of actin polymerization with cytochalasin D caused the actin cytoskeleton to separate from the cell margin and undergo a finite retrograde retraction. In contrast, inhibition of myosin II function either with the wide-spectrum protein kinase inhibitor staurosporine or the myosin light chain kinase-specific inhibitor KT5926 stopped flow in the cell center, whereas normal retrograde flow continued at the cell periphery. These differential results suggest that the mechanism of retrograde flow has two, spatially segregated components. We propose a "push-pull" mechanism in which actin polymerization drives flow at the cell periphery, whereas myosin II provides the tension on the actin cytoskeleton necessary for flow in the cell interior.
Mol Biol Cell 1999 Dec
PMID:Two components of actin-based retrograde flow in sea urchin coelomocytes. 1058 44

The purpose of this study was to examine whether the nitric oxide donor S-nitrosoglutathione (GSNO) relaxes canine tracheal smooth muscle (CTSM) strips by decreasing Ca(2+) sensitivity [i.e., the amount of force for a given intracellular Ca(2+) concentration ([Ca(2+)](i))]. We further investigated whether GSNO decreases Ca(2+) sensitivity by altering the relationship between regulatory myosin light chain (rMLC) phosphorylation and [Ca(2+)](i) and the relationship between force and rMLC phosphorylation. GSNO (100 microM) relaxed intact CTSM strips contracted with 45 mM KCl by decreasing Ca(2+) sensitivity in comparison to control strips without significantly decreasing [Ca(2+)](i). GSNO reduced the amount of rMLC phosphorylation for a given [Ca(2+)](i) but did not affect the relationship between isometric force and rMLC phosphorylation. These results show that in CTSM strips contracted with KCl, GSNO decreases Ca(2+) sensitivity by affecting the level of rMLC phosphorylation for a given [Ca(2+)](i), suggesting that myosin light chain kinase is inhibited or that smooth muscle protein phosphatases are activated by GSNO.
Am J Physiol Lung Cell Mol Physiol 2000 Mar
PMID:S-nitrosoglutathione-induced decrease in calcium sensitivity of airway smooth muscle. 1071 May 24

The vertebrate genetic locus, coding for a Ca2+/calmodulin-dependent enzyme myosin light chain kinase (MLCK), the key regulator of smooth muscle contraction and cell motility, reveals a complex organization. Two MLCK isoforms are encoded by the MLCK genetic locus. Recently identified M(r) 210 kDa MLCK contains a sequence of smooth muscle/non-muscle M(r) 108 kDa MLCK and has an additional N-terminal sequence (Watterson et al., 1995. FEBS Lett. 373 : 217). A gene for an independently expressed non-kinase product KRP (telokin) is located within the MLCK gene (Collinge et al., 1992. Mol. Cell. Biol. 12 : 2359). KRP binds to and regulates the structure of myosin filaments (Shirinsky et al., 1993. J. Biol. Chem. 268 : 16578). Here we compared biochemical properties of MLCK-210 and MLCK-108 and studied intracellular localization of MLCK-210. MLCK-210 was isolated from extract of chicken aorta by immunoprecipitation using specific antibody and biochemically analysed in vitro. MLCK-210 phosphorylated myosin regulatory light chain and heavy meromyosin. The Ca(2+)-dependence and specific activity of MLCK-210 were similar to that of MLCK-108 from turkey gizzard. Using sedimentation assay we demonstrated that MLCK-210 as well as MLCK-108 binds to both actin and myosin filaments. MLCK-210 was localized in smooth muscle cell layers of aortic wall and was found to co-localize with microfilaments in cultured aortic smooth muscle cells.
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PMID:[Functional properties and intracellular localization of high molecular weight isoforms of ligh chain myosin kinase]. 1080 49

Elevated concentrations of cyclic AMP (cAMP) in the human myometrium may promote uterine quiescence during pregnancy by protein kinase A (PKA)-mediated phosphorylation and subsequent inactivation of myosin light-chain kinase, as well as by the phosphorylation and activation of cAMP-dependent transcription factors. In this context, we show that the altered expression of cAMP response-element binding protein (CREB), cAMP response-element modulator protein (CREM) and activating transcription factor 2 (ATF2) are implicated in the maintenance of myometrial quiescence during fetal maturation and the switch to uterine activation at term. Using electrophoretic mobility shift and super shift assays, as well as immunoblotting of paired myometrial tissue samples from non-pregnant, pregnant non-labouring and spontaneous labouring women, we defined the patterns of expression of various isoforms of these proteins in the human uterus. Here, we report spatio-temporal changes in the expression of a 43 kDa form of CREB, a 28 kDa CREM-like protein, and a novel 28 kDa ATF2-like protein which are differentially expressed, depending on the gestational state of the uterus. Changes in the pattern of expression of these potent transcription factors may have an important role in the control of uterine activity throughout pregnancy.
Mol Hum Reprod 2000 Jul
PMID:Expression of the cyclic AMP-dependent transcription factors, CREB, CREM and ATF2, in the human myometrium during pregnancy and labour. 1087 53

Intracellular pH regulation in primary cultures of neonatal cardiac myocytes has been characterized. Myocytes were exposed to hyperosmolar solutions to examine the effects on pH regulation by the Na+/H+ exchanger. Exposure to 100 mM NaCl, sorbitol, N-methyl-D-glucamine, or choline chloride all caused significant increases in steady state pHi in myocytes. Omission of extracellular calcium or administration of calmodulin antagonists reduced the osmotic activation of the exchanger. The myosin light-chain inhibitor ML-7 completely blocked osmotic activation of the exchanger suggesting that myosin light-chain kinase is involved in osmotic activation of the exchanger in the myocardium. The calmodulin-dependent protein kinase II inhibitor KN-93 inhibited the rate of recovery from an acute acid load as did trifluoperazine (TFP) and the calmodulin blocker W7, [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide]. Addition of the calcium ionophore ionomycin caused a large increase in resting pHi in isolated myocytes. However, this effect was largely resistant to HMA (5-(N,N-hexamethylene)-amiloride) indicating that an alternative mechanism of pHi regulation is responsible. The results demonstrate that the Na+/H+ exchanger of the neonatal myocardium is responsive to calcium and osmotically responsive pathways and that myosin light-chain kinase is a key protein involved in mediating the osmotic response.
J Mol Cell Cardiol 2000 Jun
PMID:Calcium and osmotic regulation of the Na+/H+ exchanger in neonatal ventricular myocytes. 1088 47

Members of the titin/myosin light chain kinase family play an essential role in the organization of the actin/myosin cytoskeleton, especially in sarcomere assembly and function. In Drosophila melanogaster, projectin is so far the only member of this family for which a transcription unit has been characterized. The locus of another member of this family, a protein related to Myosin light chain kinase, was also identified. The cDNA and genomic sequences published explain only the shorter transcripts expressed by this locus. Here, we report the complete molecular characterization of this transcription unit, which spans 38 kb, includes 33 exons and accounts for transcripts up to 25 kb in length. This transcription unit contains both the largest exon (12,005 nt) and the largest coding region (25,213 nt) reported so far for Drosophila. This transcription unit features both internal promoters and internal polyadenylation signals, which enable it to express seven different transcripts, ranging from 3.3 to 25 kb in size. The latter encodes a huge, titin-like, 926 kDa kinase that features two large PEVK-rich repeats, 32 immunoglobulin and two fibronectin type-III domains, which we designate stretchin-MLCK. In addition, the 3' end of the stretchin-MLCK transcription unit expresses shorter transcripts that encode 86 to 165 kDa isoforms of stretchin-MLCK that are analogous to vertebrate Myosin light chain kinases. Similarly, the 5' end of the Stretchin-Mlck transcription unit can also express transcripts encoding kettin and Unc-89-like isoforms, which share no sequences with the MLCK-like transcripts. Thus, this locus can be viewed as a single transcription unit, Stretchin-Mlck (genetic abbreviation Strn-Mlck), that expresses large, composite transcripts and protein isoforms (sequences available at http://www.academicpress.com/jmb), as well as a complex of two independent transcription units, the Stretchin and Mlck transcription units (Strn and Mlck, respectively) the result of a "gene fission" event, that encode independent transcripts and proteins with distinct structural and enzymatic functions.
J Mol Biol 2000 Jul 21
PMID:Drosophila stretchin-MLCK is a novel member of the Titin/Myosin light chain kinase family. 1089 Dec 86

Reactive oxygen species (ROS) generated by activated leukocytes play an important role in the disruption of endothelial cell (EC) integrity, leading to barrier dysfunction and pulmonary edema. Although ROS modulate cell signaling, information remains limited regarding the mechanism(s) of ROS-induced EC barrier dysfunction. We utilized diperoxovanadate (DPV) as a model agent to explore the role of tyrosine phosphorylation in the regulation of EC barrier function. DPV disrupted EC barrier function in a dose-dependent manner. Tyrosine kinase inhibitors, genistein, and PP-2, a specific inhibitor of Src, reduced the DPV-mediated barrier dysfunction. Consistent with these results, DPV-induced Src activation was attenuated by PP-2. Furthermore, DPV increased the association of Src with cortactin and myosin light chain kinase, indicating their potential role as cytoskeletal targets for Src. Transient overexpression of either wild-type Src or a constitutively active Src mutant potentiated the DPV-mediated decline in barrier dysfunction, whereas a dominant negative Src mutant attenuated the response. These studies provide the first direct evidence for Src involvement in DPV-induced EC barrier dysfunction.
Am J Physiol Lung Cell Mol Physiol 2000 Sep
PMID:Involvement of c-Src in diperoxovanadate-induced endothelial cell barrier dysfunction. 1095 17


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