Functional properties and intracellular localization of high molecular weight isoforms of ligh chain myosin kinase

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.     

Analysis of the kinase-related protein gene found at human chromosome 3q21 in a multi-gene cluster: organization, expression, alternative splicing, and polymorphic marker.

We report the amino acid sequence, genomic organization, tissue expression, and alternative splice patterns for the human kinase related protein (KRP) gene, as well as the discovery of a new CA repeat sequence polymorphic marker in an upstream intron of the myosin light chain kinase (MLCK) gene. The KRP/MLCK genetic locus is a prototype for a recently discovered paradigm in which an independently regulated gene for a non-enzymic protein is embedded within a larger gene for a signal transduction enzyme, and both classes of proteins are involved in the regulation of the same cellular structure. The MLCK/KRP gene cluster has been found only in higher vertebrates and is localized to human chromosome 3q21. The determination of the human KRP amino acid sequence through cDNA sequence analysis and its comparison to the exon/intron organization of the human KRP gene revealed an alternative splice pattern at the start of KRP exon 2, resulting in the insertion of a single glutamic acid in the middle of the protein. Examination of tissue distribution using Northern blot analysis revealed that the human expression pattern is more similar to the well-characterized chicken KRP gene expression pattern than to rodent or rabbit. Unexpected differences of the human gene from other species is the apparent expression of the human gene products in adult cardiac muscle, an observation that was pursued further by the production of a site-directed antiserum and immunohistochemistry analysis. The results reported here provide insight into the conserved and variable features of this late evolving genetic paradigm, raise new questions about the molecular aspects of cardiac muscle regulation, and provide tools needed for future clinical studies. The comparative analysis of the MLCK/KRP locus, combined with the recent discovery of a similar genomic relationship among other signal transduction proteins, suggest a diverse distribution of this theme among signal transduction systems in higher vertebrate genomes and indicate the utility of comparative genomics in revealing late evolving genetic paradigms.     

Myosin light chain kinase colocalizes with nonmuscle myosin IIB in myofibril precursors and sarcomeric Z-lines of cardiomyocytes

Myosin light chain kinase (MLCK) is a key regulator of various forms of cell motility involving actin and myosin II. MLCK is widely present in vertebrate tissues including the myocardium. However, the role of MLCK in cardiomyocyte function is not known. Previous attempts to gain insight into possible roles and identify potential molecular partners were disappointing and equivocal due to cross reactivity of early antibodies with striated muscle MLCK, which has a different genetic locus and a divergent amino acid sequence from the above mentioned enzyme. Using an immunofluorescence approach and a panel of antibodies directed against MLCK, cytoskeletal, and sarcomeric proteins, we localized MLCK to myofibril precursors and Z-lines of sarcomeres in embryonic and adult cardiomyocytes. The same structures contained nonmuscle myosin IIB implicating this protein as a possible target of MLCK. Our results suggest a role for MLCK in cardiomyocyte differentiation and contraction through regulation of nonmuscle myosin IIB.     

Differences in phorbol-dependent phosphorylation of regulatory proteins and contraction of phasic and tonic smooth muscle

Smooth muscles are divided into slowly contracting tonic and relatively fast phasic muscles. In both cases Ca2+ is a key mediator of the contractile response. However, the appearance of a tonic component during sphincter or arterial muscle contraction and its absence in contracting visceral smooth muscle is characteristic of their difference. We have found that in chicken tissues phorbol 12,13-dibutyrate (PDBu) induces a sustained contraction in carotid arterial muscle, but provokes no contraction in phasic gizzard smooth muscle. Next we were aimed to find differences in PDBu-induced phosphorylation of the key proteins involved in regulation of smooth muscle contraction, i.e. caldesmon, myosin light chain kinase (MLCK), and the myosin light chain kinase-related protein (KRP, also known as telokin). Two correlative differences were observed. 1. PDBu stimulated phosphorylation of MLCK in tonic smooth muscle and had no effect on the level of MLCK phosphorylation in phasic muscle. Phosphopeptide mapping suggests the involvement of mitogen-activated protein (MAP) kinases in phosphorylation of MLCK in situ. 2. PDBu induced phosphorylation of MAP-kinase sites in caldesmon in both types of smooth muscle, but this phosphorylation had no significant effect on caldesmon functional activity in vitro. For the first time we have shown that in gizzard PDBu also stimulates a yet unknown transitory caldesmon-kinase different from protein kinase, C, Ca2+/calmodulin-dependent kinase II and casein kinase CK2. 3. No significant difference was found in the kinetics of PDBu-dependent phosphorylation of KRP in tonic and phasic smooth muscles. KRP was also demonstrated to be a major phosphoprotein in smooth muscle phosphorylated in vivo at several sites located within its N-terminal sequence. Protein kinases able to phosphorylate these sites were identified in vitro. Among them, MAP-kinase was suggested to phosphorylate a serine residue homologous to that phosphorylated in MLCK. 4. p42erk2 and p38 MAP-kinases were found in phasic and tonic smooth muscles. Both were responsive to PDBu in cultured chicken aortic smooth muscle cells, and their role in phosphorylation of MLCK and low molecular weight isoform of caldesmon was evaluated.     

Novel Phosphospecific Antibodies for Monitoring Phosphorylation of Proteins Encoded by the Myosin Light Chain Kinase Genetic Locus

Myosin light chain kinase (MLCK) and the kinase-related protein (KRP), also known as telokin, are the major independent protein products of the smooth muscle/non-muscle MLCK genetic locus. They share a common C-terminal part and major sites phosphorylated in vivo. Whereas MLCK is critically involved in myosin activation and contraction initiation in smooth muscle, KRP is thought to antagonize MLCK and to exert relaxation activity. Phosphorylation controls the MLCK and KRP activities. We generated two phosphorylation and site-specific antibodies to individually monitor levels of MLCK and KRP phosphorylation on critical sites. We quantified the level of KRP phosphorylation in smooth muscle before and after an increase in intracellular free Ca2+ and stimulation of adenylate cyclase, protein kinase C, and mitogen-activated protein kinases (MAP-kinases). Forskolin and phorbol-12,13-dibutyrate increased KRP phosphorylation at Ser13 from 25 to 100% but did not produce contraction in rat ileum. The level of Ser13 phosphorylation was not altered during Ca2+-dependent contraction evoked by KCl depolarization or carbachol, but subsequently increased to maximum during forskolin-induced relaxation. These data suggest that several intracellular signaling pathways control phosphorylation of KRP on Ser13 in smooth muscle and thus may contribute to relaxation. In contrast, phosphorylation level of Ser19 of KRP increased only slightly (from 30 to 40-45%) and only in response to MAP-kinase activation, arguing against its regulatory function in smooth muscle.     

Localization and activity of myosin light chain kinase isoforms during the cell cycle

Phosphorylation on Ser 19 of the myosin II regulatory light chain by myosin light chain kinase (MLCK) regulates actomyosin contractility in smooth muscle and vertebrate nonmuscle cells. The smooth/nonmuscle MLCK gene locus produces two kinases, a high molecular weight isoform (long MLCK) and a low molecular weight isoform (short MLCK), that are differentially expressed in smooth and nonmuscle tissues. To study the relative localization of the MLCK isoforms in cultured nonmuscle cells and to determine the spatial and temporal dynamics of MLCK localization during mitosis, we constructed green fluorescent protein fusions of the long and short MLCKs. In interphase cells, localization of the long MLCK to stress fibers is mediated by five DXRXXL motifs, which span the junction of the NH(2)-terminal extension and the short MLCK. In contrast, localization of the long MLCK to the cleavage furrow in dividing cells requires the five DXRXXL motifs as well as additional amino acid sequences present in the NH(2)-terminal extension. Thus, it appears that nonmuscle cells utilize different mechanisms for targeting the long MLCK to actomyosin structures during interphase and mitosis. Further studies have shown that the long MLCK has twofold lower kinase activity in early mitosis than in interphase or in the early stages of postmitotic spreading. These findings suggest a model in which MLCK and the myosin II phosphatase (Totsukawa, G., Y. Yamakita, S. Yamashiro, H. Hosoya, D.J. Hartshorne, and F. Matsumura. 1999. J. Cell Biol. 144:735-744) act cooperatively to regulate the level of Ser 19-phosphorylated myosin II during mitosis and initiate cytokinesis through the activation of myosin II motor activity.     

Use of DNA sequence and mutant analyses and antisense oligodeoxynucleotides to examine the molecular basis of nonmuscle myosin light chain kinase autoinhibition, calmodulin recognition, and activity

The first primary structure for a nonmuscle myosin light chain kinase (nmMLCK) has been determined by elucidation of the cDNA sequence encoding the protein kinase from chicken embryo fibroblasts, and insight into the molecular mechanism of calmodulin (CaM) recognition and activation has been obtained by the use of site-specific mutagenesis and suppressor mutant analysis. Treatment of chicken and mouse fibroblasts with antisense oligodeoxynucleotides based on the cDNA sequence results in an apparent decrease in MLCK levels, an altered morphology reminiscent of that seen in v-src-transformed cells, and a possible effect on cell proliferation. nmMLCK is distinct from and larger than smooth muscle MLCK (smMLCK), although their extended DNA sequence identity is suggestive of a close genetic relationship not found with skeletal muscle MLCK. The analysis of 20 mutant MLCKs indicates that the autoinhibitory and CaM recognition activities are centered in distinct but functionally coupled amino acid sequences (residues 1,068-1,080 and 1,082-1,101, respectively). Analysis of enzyme chimeras, random mutations, inverted sequences, and point mutations in the 1,082-1,101 region demonstrates its functional importance for CaM recognition but not autoinhibition. In contrast, certain mutations in the 1,068-1,080 region result in a constitutively active MLCK that still binds CaM. These results suggest that CaM/protein kinase complexes use similar structural themes to transduce calcium signals into selective biological responses, demonstrate a direct link between nmMLCK and non-muscle cell function, and provide a firm basis for genetic studies and analyses of how nmMLCK is involved in development and cell proliferation.     

A novel regulatory protein that affects the functions of caldesmon and myosin light chain kinase.

A caldesmon (CaD)-binding protein of about 65 kDa (by SDS-PAGE) was purified from smooth muscle of chicken gizzard. The 65-kDa protein prevented the inhibitory effect of CaD on the ATP-dependent interaction between actin and myosin. Unlike the case with calmodulin (CaM), Ca2+ was not required for this effect. As reported in the preceding communication, myosin light chain kinase (MLCK), another well characterized protein that binds CaM, has CaD-like activity that modulates the interaction by binding to actin. The 65-kDa protein was also effective in relieving the modulation, while leaving unaffected the kinase activity that phosphorylates the light chain of smooth muscle myosin.     

AMP-activated protein kinase phosphorylates and desensitizes smooth muscle myosin light chain kinase

Smooth muscle contraction is initiated by a rise in intracellular calcium, leading to activation of smooth muscle myosin light chain kinase (MLCK) via calcium/calmodulin (CaM). Activated MLCK then phosphorylates the regulatory myosin light chains, triggering cross-bridge cycling and contraction. Here, we show that MLCK is a substrate of AMP-activated protein kinase (AMPK). The phosphorylation site in chicken MLCK was identified by mass spectrometry to be located in the CaM-binding domain at Ser(815). Phosphorylation by AMPK desensitized MLCK by increasing the concentration of CaM required for half-maximal activation. In primary cultures of rat aortic smooth muscle cells, vasoconstrictors activated AMPK in a calcium-dependent manner via CaM-dependent protein kinase kinase-beta, a known upstream kinase of AMPK. Indeed, vasoconstrictor-induced AMPK activation was abrogated by the STO-609 CaM-dependent protein kinase kinase-beta inhibitor. Myosin light chain phosphorylation was increased under these conditions, suggesting that contraction would be potentiated by ablation of AMPK. Indeed, in aortic rings from mice in which alpha1, the major catalytic subunit isoform in arterial smooth muscle, had been deleted, KCl- or phenylephrine-induced contraction was increased. The findings suggest that AMPK attenuates contraction by phosphorylating and inactivating MLCK. This might contribute to reduced ATP turnover in the tonic phase of smooth muscle contraction.     

Distribution of calmodulin and calmodulin-binding proteins in bovine pituitary: association of myosin light chain kinase with pituitary secretory granule membranes

Calcium is necessary for secretion of pituitary hormones. Many of the biological effects of Ca²⁺ are mediated by the Ca²⁺-binding protein calmodulin (CaM), which interacts specifically with proteins regulated by the Ca²⁺-CaM complex. One of these proteins is myosin light chain kinase (MLCK), a Ca²⁺-calmodulin dependent enzyme that phosphorylates the regulatory light chains of myosin, and has been implicated in motile processes in both muscle and non-muscle tissues. We determined the content and distribution of CaM and CaM-binding proteins in bovine pituitary homogenates, and subcellular fractions including secretory granules and secretory granule membranes. CaM measured by radioimmunoassay was found in each fraction; although approximately one-half was in the cytosolic fraction, CaM was also associated with the plasma membrane and secretory granule fractions. CaM-binding proteins were identified by an ²⁵¹-CaM gel overlay technique and quantitated by densitometric analysis of the autoradiograms. Pituitary homogenates contained nine major CaM-binding proteins of 146, 131, 90, 64, 58, 56, 52, 31 and 22 kilodaltons (kDa). Binding to all the bands was specific, Cat+-sensitive, and displaceable with excess unlabeled CaM. Severe heat treatment (100°C, 15 min), which results in a 75% reduction in phosphodiesterase activation by CaM, markedly decreased ²⁵¹I-CaM binding to all protein bands. Secretory granule membranes showed enhancement for CaM-binding proteins with molecular weights of 184, 146, 131, 90, and 52000. A specific, affinity purified antibody to chicken gizzard MLCK bound to the 146 kDa band in homogenates, centrifugal subcellular fractions, and secretory granule membranes. No such binding was associated with the granule contents. The enrichment of MLCK and other CaM-binding proteins in pituitary secretory granule membranes suggests a possible role for CaM and/or CaM-binding proteins in granule membrane function and possibly exocytosis.     

Vectorial activation of smooth muscle myosin filaments and its modulation by telokin

Smooth muscle myosin copurifies with myosin light chain kinase (MLCK) and calmodulin (CaM) as well as with variable amounts of myosin phosphatase. Therefore, myosin filaments formed in vitro also contain relatively high levels of these enzymes. Thus these filaments may be considered to be native-like because they are similar to those expected to exist in vivo. These endogenous enzymes are present at high concentrations relative to myosin, sufficient for rapid phosphorylation and dephosphorylation of the filaments at rates comparable to those observed for contraction and relaxation in intact muscle strips. The phosphorylation by MLCK/CaM complex appears to exhibit some directionality and is not governed by a random diffusional process. For the mixtures of myosin filaments with and without the endogenous MLCK/CaM complex, the complex preferentially phosphorylates its own parent filament at a higher rate than the neighboring filaments. This selective or vectorial-like activation is lost or absent when myosin filaments are dissolved at high ionic strength. Similar vectorial-like activation is exhibited by the reconstituted filament suspensions, but the soluble systems composed of isolated regulatory light chain or soluble myosin head subfragments exhibit normal diffusional kinetic behavior. At physiological concentrations, kinase related protein (telokin) effectively modulates the activation process by reducing the phosphorylation rate of the filaments without affecting the overall phosphorylation level. This results from telokin-induced liberation of the active MLCK/CaM complex from the filaments, so that the latter can also activate the neighboring filaments via a slower diffusional process. When this complex is bound at insufficient levels, this actually results in acceleration of the initial phosphorylation rates. In short, I suggest that in smooth muscle, telokin plays a chaperone role for myosin and its filaments.     

Quantitative measurements of Ca(2+)/calmodulin binding and activation of myosin light chain kinase in cells

Myosin II regulatory light chain (RLC) phosphorylation by Ca(2+)/calmodulin (CaM)-dependent myosin light chain kinase (MLCK) is implicated in many cellular actin cytoskeletal functions. We examined MLCK activation quantitatively with a fluorescent biosensor MLCK where Ca(2+)-dependent increases in kinase activity were coincident with decreases in fluorescence resonance energy transfer (FRET) in vitro. In cells stably transfected with CaM sensor MLCK, increasing [Ca(2+)](i) increased MLCK activation and RLC phosphorylation coincidently. There was no evidence for CaM binding but not activating MLCK at low [Ca(2+)](i). At saturating [Ca(2+)](i) MLCK was not fully activated probably due to limited availability of cellular Ca(2+)/CaM.     

Alterations in myocardial ultrastructure and protein expression after a single injection of isoproterenol

Immunochemical and electron microscopic characterization of rat myocardium was conducted 2 h and 3 weeks after a single injection of isoproterenol in rats. The relative content of several myospecific proteins (KRP – kinase-related protein, desmin), cytoskeletal proteins (tubulin, vinculin, myosin light chain kinase – MLCK) and extracellular matrix protein fibronectin was determined by immunoblotting. Two hours after injection of 50 mg/kg isoproterenol a destruction of some cardiomyocytes, contracture of myofibrils and mild edema of intercellular space was observed. The content of all the studied proteins except KRP decreased below control levels. This situation sustained 3 weeks after injection and paralleled alterations in cardiomyocyte ultrastructure. Areas of myofibrillar contracture and lysis were noted, glycogen granules were sparse; mitochondria contained arrow-like inclusions that are characteristic for calcium overload, also huge mitochondria contacting each other by specialized intermitochondrial contacts were detected. Clumps of unripe elastic fibers in enlarged intercellular space were combined with increased deposition of collagens type I and III forming areas of fibrosis. The smaller dosage of isoproterenol (10 mg/kg) rendered no significant damage in the acute postinjection period but 3 weeks later it induced the thickening of extracellular matrix around cardiac cells and the increase in KRP and tubulin content by 26 and 32%, correspondingly. MLCK levels remained depressed throughout the experiment. The rise in KRP expression was also observed after the addition of isoproterenol to cultured chicken embryo cardiomyocytes. Obtained results indicate that even a single injection of isoproterenol creates long lasting structural alterations in cardiac muscle accompanied by the increased expression of extracellular matrix proteins and several sarcoplasmic proteins apparently involved in hypertrophic response of cardiomyocytes.     

The heterodimer calmodulin: myosin light-chain kinase as a prototype vertebrate calcium signal transduction complex.

The heterodimer complex of calmodulin (CaM) and the protein kinase catalytic subunit of myosin light chain kinase from vertebrate smooth muscle and non-muscle tissues (sm/nmMLCK) is one of the most extensively characterized CaM-regulated enzyme complexes and it has an established in vivo role in the transduction of calcium signals into biological responses. We have used a combination of approaches to the study of CaM and sm/nmMLCK in order to derive initial insight into the key features of each protein and of the CaM-MLCK heterodimeric complex that are involved in protein-protein and calcium-protein recognition and regulation of enzyme activity. On-going studies are described here that include site-specific mutagenesis, fluorescence spectroscopy, enzymology and peptide analog analysis. These and previous results indicate that: (1), both electrostatic and hydrophobic features are important in the functionally correct interactions between CaM and MLCK; (2), even the interactions between CaM and peptide analogs of the CaM binding site of MLCK are heterogeneous and non-trivial in nature; (3), amino-acid residues that have been conserved in CaM across millions of years of evolution and that are conserved in CaMs with quantitative MLCK activator activity can be mutated without any detectable effect on activity and (4), structures different from the prototypical EF-hand domain of CaM can have similar calcium-binding activity in the presence of a CaM binding structure.     

Functional interactions between smooth muscle myosin light chain kinase and calmodulin

Calmodulin (CaM) binding by turkey gizzard myosin light chain kinase (MLCK) causes subtle changes in the fluorescence emission and polarization excitation spectra of the enzyme. Fluorescence experiments using 9-anthroyl-choline (9AC), which competes with ATP in binding, demonstrate mutually stabilizing interactions between the CaM and ATP binding sites corresponding to delta G = -0.6 to -0.7 kcal/mol. Fluorescence titrations in the presence of 9AC or 5,5'-bis[8-(phenylamino)-1-naphthalenesulfonate] confirm the stoichiometry of 1 mol of CaM/MLCK. Phosphorylation of MLCK has no effect on either the protein fluorescence or the binding of ATP and 9AC. The dissociation constant for the MLCL-CaM complex is increased approximately 500-fold on phosphorylation. Values of Kd for the phosphorylated enzyme range from 0.5 to 1.1 microM in 0.2 N KCl, pH 7.3, 25 degrees C. We showed competition between MLCK and other CaM binding proteins and peptides by using both fluorescence and catalytic activity measurements. Competition for CaM occurs with ACTH, beta-endorphin, substance P, glucagon, poly(L-arginine), myelin basic protein, troponin I, and histone H2A. Phosphorylation of the last three proteins by the adenosine cyclic 3',5'-phosphate dependent protein kinase diminishes their ability to compete. Phosphorylation of MLCK by the protein kinase gives 0.95 +/- 0.04 and 2.2 +/- 0.4 mol of incorporated 32P in the presence and absence of CaM, respectively. These stoichiometries agree with those recently reported [Conti, M. A. & Adelstein, R. S. (1981) J. Biol. Chem. 256, 3178].     

Involvement of calmodulin and myosin light chain kinase in activation of mTRPC5 expressed in HEK cells

The classic type of transient receptor potential channel (TRPC) is a molecular candidate for Ca(2+)-permeable cation channels in mammalian cells. Because TRPC channels have calmodulin (CaM) binding sites at their COOH termini, we investigated the effect of CaM on mTRPC5. TRPC5 was initially activated by muscarinic stimulation with 50 microM carbachol and then decayed rapidly even in the presence of carbachol. Intracellular CaM (150 microg/ml) increased the amplitude of mTRPC5 current activated by muscarinic stimulation. CaM antagonists (W-7 and calmidazolium) inhibited mTRPC5 currents when they were applied during the activation of mTRPC5. Pretreatment of W-7 and calmidazolium also inhibited the activation of mTRPC5 current. Inhibitors of myosin light chain kinase (MLCK) inhibited the activation of mTRPC5 currents, whereas inhibitors of CaM-dependent protein kinase II did not. Small interfering RNA against cardiac type MLCK also inhibited the activation of mTRPC5 currents. However, inhibitors of CaM or MLCK did not show any effect on GTPgammaS-induced currents. Application of both Rho kinase inhibitor and MLCK inhibitor inhibited GTPgammaS-induced currents. We conclude that CaM and MLCK modulates the activation process of mTRPC5.     

ZIP kinase identified as a novel myosin regulatory light chain kinase in HeLa cells.

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.     

Mitosis-specific phosphorylation of myosin light chain kinase

Cell cytosol preparations from mitotic HeLa cells exhibit a kinase activity that phosphorylates myosin light chain kinase (MLCK). This MLCK kinase activity is apparently distinct from the known MLCK kinases, including cAMP-dependent protein kinase, cGMP-dependent protein kinase, Ca(2+)-activated phospholipid-dependent protein kinase, or Ca(2+)-calmodulin-dependent protein kinase II, based on the following criteria. First, the MLCK kinase activity of mitotic cells does not respond to a variety of characteristic activators or inhibitors of these known kinases. Second, one- and two-dimensional peptide maps have revealed that the site of phosphorylation by the MLCK kinase of mitotic cells differs from those by these known kinases. The mitotic MLCK kinase phosphorylates MLCK at a threonine residue at a ratio of up to 1 mol of phosphate/mol of chicken gizzard MLCK. The MLCK kinase is mitosis-specific because mitotic cell extracts show much higher phosphorylation activity than nonmitotic cell extracts.     

220- and 130-kDa MLCKs have distinct tissue distributions and intracellular localization patterns

To better understand thedistinct functional roles of the 220- and 130-kDa forms of myosin lightchain kinase (MLCK), expression and intracellular localization weredetermined during development and in adult mouse tissues. Northernblot, Western blot, and histochemical studies show that the 220-kDaMLCK is widely expressed during development as well as in several adultsmooth muscle and nonmuscle tissues. The 130-kDa MLCK is highlyexpressed in all adult tissues examined and is also detectable duringembryonic development. Colocalization studies examining thedistribution of 130- and 220-kDa mouse MLCKs revealed that the 130-kDaMLCK colocalizes with nonmuscle myosin IIA but not with myosin IIB orF-actin. In contrast, the 220-kDa MLCK did not colocalize with eithernonmuscle myosin II isoform but instead colocalizes with thickinterconnected bundles of F-actin. These results suggest that in vivo,the physiological functions of the 220- and 130-kDa MLCKs are likely tobe regulated by their intracellular trafficking and distribution.