Phosphorylation Regulates Interaction of 210-kDa Myosin Light Chain Kinase N-terminal Domain with Actin Cytoskeleton.

High molecular weight myosin light chain kinase (MLCK210) is a multifunctional protein involved in myosin II activation and integration of cytoskeletal components in cells. MLCK210 possesses actin-binding regions both in the central part of the molecule and in its N-terminal tail domain. In HeLa cells, mitotic protein kinase Aurora B was suggested to phosphorylate MLCK210 N-terminal tail at serine residues (Dulyaninova, N. G., and Bresnick, A. R. (2004) Exp. Cell Res., 299, 303-314), but the functional significance of the phosphorylation was not established. We report here that in vitro, the N-terminal actin-binding domain of MLCK210 is located within residues 27-157 (N27-157, avian MLCK210 sequence) and is phosphorylated by cAMP-dependent protein kinase (PKA) and Aurora B at serine residues 140/149 leading to a decrease in N27-157 binding to actin. The same residues are phosphorylated in a PKA-dependent manner in transfected HeLa cells. Further, in transfected cells, phosphomimetic mutants of N27-157 showed reduced association with the detergent-stable cytoskeleton, whereas in vitro, the single S149D mutation reduced N27-157 association with F-actin to a similar extent as that achieved by N27-157 phosphorylation. Altogether, our results indicate that phosphorylation of MLCK210 at distinct serine residues, mainly at S149, attenuates the interaction of MLCK210 N-terminus with the actin cytoskeleton and might serve to regulate MLCK210 microfilament cross-linking activity in cells.

[Penetrating peptide inhibitor of the myosin light chain kinase suppresses hyperpermeability of vascular endothelium].

Nonapeptide H-Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2 corresponding to a modified sequence of autoinhibitory region of myosin light chain kinase (MLCK) was synthesized from L-amino acids and from D-amino acids. Using nuclear magnetic resonance spectroscopy it has been demonstrated that D-peptide is significantly more stable in human blood plasma than its L-enantiomer. D-peptide accumulated in cultured human umbilical vein endothelial cells suppressed development of hyperpermeability in endothelial monolayer induced by thrombin addition. Following intravenous administration D-peptide decreased the extent of lung oedema in rats induced by infusion of oleic acid in bloodstream. Thus, the peptide molecules based on an autoinhibitory peptide of MLCK may serve as a prototype for development of a novel antioedematous drugs that directly affect the MLCK-dependent motile processes in vascular endothelium.

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.

Mechanism of glial activation by S100B: involvement of the transcription factor NFkappaB.

Compelling evidence links chronic activation of glia and the subsequent cycle of neuroinflammation and neuronal dysfunction to the progression of neurodegeneration in disorders such as Alzheimer's disease (AD). S100B, a glial-derived cytokine, is significantly elevated in the brains of AD patients and high concentrations of S100B are believed to be detrimental to brain function. As a first step toward elucidating the mechanisms by which S100B might be serving this detrimental role, we examined the mechanisms by which S100B stimulates glial inducible nitric oxide synthase (iNOS), an oxidative stress related enzyme that has been linked to neuropathology through the production of neurotoxic peroxynitrite. We report here that S100B stimulates iNOS in rat primary cortical astrocytes through a signal transduction pathway that involves activation of the transcription factor NFkappaB. NFkappaB activation was demonstrated by nuclear translocation of the p65 NFkappaB subunit, stimulation of NFkappaB-specific DNA binding activity, and stimulation of NFkappaB-dependent transcriptional activity. Furthermore, S100B-induced iNOS promoter activation was inhibited upon mutation of the NFkappaB response element in the promoter, and transfection of cells with an NFkappaB inhibitor blocked S100B-induced iNOS promoter activation and nitric oxide production. These studies define a signal transduction pathway by which S100B activation of glia could participate in the generation of oxidative stress in the brain.

Ligand modulation of glial activation: cell permeable, small molecule inhibitors of serine-threonine protein kinases can block induction of interleukin 1 beta and nitric oxide synthase II.

Activated glia (astrocytes and microglia) and their associated neuroinflammatory sequelae have been linked to the disease progression of several neurodegenerative disorders, including Alzheimer's disease. We found that the experimental anti-inflammatory drug K252a, an inhibitor of calmodulin regulated protein kinases (CaMKs), can block induction of both the oxidative stress related enzyme iNOS and the proinflammatory cytokine IL-1 beta in primary cortical glial cultures and the microglial BV-2 cell line. We also found that the profile of CaMKIV and CaMKII isoforms in primary cortical glial cultures and BV-2 cells is distinct from that found in neurons. Knowledge of cellular mechanisms and high throughput screens of a pharmacologically focused chemical library allowed the discovery of novel pyridazine-based compounds that are cell permeable ligand modulators of gene regulating protein kinases involved in the induction of iNOS and IL-1 beta in activated glia. Pyridazine-based compounds are attractive for the development of new therapeutics due to the retention of the remarkable pharmacological properties of K252a and related indolocarbazole alkaloids, and presence of enhanced functional selectivity in a comparatively simple structure amenable to diverse synthetic chemistries.

Crystal structures of the catalytic domain of human protein kinase associated with apoptosis and tumor suppression.

We have determined X-ray crystal structures with up to 1.5 A resolution of the catalytic domain of death-associated protein kinase (DAPK), the first described member of a novel family of pro-apoptotic and tumor-suppressive serine/threonine kinases. The geometry of the active site was studied in the apo form, in a complex with nonhydrolyzable AMPPnP and in a ternary complex consisting of kinase, AMPPnP and either Mg2+ or Mn2+. The structures revealed a previously undescribed water-mediated stabilization of the interaction between the lysine that is conserved in protein kinases and the beta- and gamma-phosphates of ATP, as well as conformational changes at the active site upon ion binding. Comparison between these structures and nucleotide triphosphate complexes of several other kinases disclosed a number of unique features of the DAPK catalytic domain, among which is a highly ordered basic loop in the N-terminal domain that may participate in enzyme regulation.

A protein kinase associated with apoptosis and tumor suppression: structure, activity, and discovery of peptide substrates.

Death-associated protein kinase (DAPK) has been implicated in apoptosis and tumor suppression, depending on cellular conditions, and associated with mechanisms of disease. However, DAPK has not been characterized as an enzyme due to the lack of protein or peptide substrates. Therefore, we determined the structure of DAPK catalytic domain, used a homology model of docked peptide substrate, and synthesized positional scanning substrate libraries in order to discover peptide substrates with K(m) values in the desired 10 microm range and to obtain knowledge about the preferences of DAPK for phosphorylation site sequences. Mutagenesis of DAPK catalytic domain at amino acids conserved among protein kinases or unique to DAPK provided a link between structure and activity. An enzyme assay for DAPK was developed and used to measure activity in adult brain and monitor protein purification based on the physical and chemical properties of the open reading frame of the DAPK cDNA. The results allow insight into substrate preferences and regulation of DAPK, provide a foundation for proteomic investigations and inhibitor discovery, and demonstrate the utility of the experimental approach, which can be extended potentially to kinase open reading frames identified by genome sequencing projects or functional genetics screens and lacking a known substrate.

Replacement of Lys-75 of calmodulin affects its interaction with smooth muscle caldesmon.

The interaction of smooth muscle caldesmon with synthetic calmodulin (SynCam) and its five mutants with replacement of Lys-75 was analyzed by means of intrinsic Trp fluorescence, zero-length crosslinking and by caldesmon-induced inhibition of actomyosin ATPase activity. SynCam and its double mutant with replacement K75P and simultaneous insertion of KGK between residues 80 and 81 have a comparably low affinity to caldesmon and the probability of crosslinking of this mutant to caldesmon was the lowest among all mutants analyzed. SynCam and its double mutant (K75P+KGK) induced nearly complete reversion of caldesmon inhibition of actomyosin ATPase activity with half-maximal reversion achieved at about 1 microM. Two mutants, K75A and K75V, with partially stabilized less positive central domain have higher affinity to caldesmon. These mutants induce 80-85% reversion of caldesmon inhibition of actomyosin ATPase and the half-maximal reversion was achieved at about 0.3-0.4 microM. Two last mutants, K75P and K75E, with distorted central domain have high affinity to caldesmon and the probability of crosslinking of K75P to caldesmon was the highest among calmodulin mutants tested. These mutants induced complete reversion of caldesmon inhibition with half-maximal effect observed at 0.3-0.4 microM. We suggest that the length, flexibility and charge of the central domain affect binding of calmodulin mutants and their ability to reverse caldesmon-induced inhibition of actomyosin ATPase activity.

Similar activation of glial cultures from different rat brain regions by neuroinflammatory stimuli and downregulation of the activation by a new class of small molecule ligands.

Activated glia (astrocytes and microglia) surrounding neuritic plaques in Alzheimer's disease (AD) overexpress an array of detrimental inflammatory molecules. Chronically activated glia and numerous inflammatory mediators in AD suggest that neuroinflammation is an integral component of the pathogenic process. However, the potential for glia from different brain regions to respond differentially to activating stimuli and inhibitors of glial activation is not well understood. As part of our goal to elucidate molecular mechanisms of glial activation, we examined the activation responses of primary cultures of glia derived from different brain regions. Neonatal rat glia from cortex, hippocampus, midbrain, brainstem, striatum, and cerebellum can be activated by a variety of stimuli (including beta-amyloid, S100B, and lipopolysaccharide), and the activation can be downregulated by a new class of small molecule, cell permeable ligands. The end points assayed included IL-1beta, iNOS, apoE and the astrocyte marker protein GFAP. The activating stimuli were able to increase the production of iNOS and IL1beta, and the ligand was able to inhibit this increase in cultures derived from the diverse brain regions. The activation and downregulation were selective, as demonstrated by lack of effect on GFAP levels and no downregulation of apoE. These results are consistent with the working hypothesis that regional differences in glial activation seen in disease and injury are reflective of the intensity, duration and repertoire of activating stimuli rather than an innate property of the glia.

Phosphorylation of kinase-related protein (telokin) in tonic and phasic smooth muscles.

KRP (telokin), an independently expressed C-terminal myosin-binding domain of smooth muscle myosin light chain kinase (MLCK), has been reported to have two related functions. First, KRP stabilizes myosin filaments (Shirinsky et al., 1993, J. Biol. Chem. 268, 16578-16583) in the presence of ATP. Secondly, KRP can modulate the level of myosin light chain phosphorylation. In this latter role, multiple mechanisms have been suggested. One hypothesis is that light chain phosphorylation is diminished by the direct competition of KRP and MLCK for myosin, resulting in a loss of contraction. Alternatively, KRP, through an unidentified mechanism, accelerates myosin light chain dephosphorylation in a manner possibly enhanced by KRP phosphorylation. Here, we demonstrate that KRP is a major phosphoprotein in smooth muscle, and use a comparative approach to investigate how its phosphorylation correlates with sustained contraction and forskolin-induced relaxation. Forskolin relaxation of precontracted artery strips caused little increase in KRP phosphorylation, while treatment with phorbol ester increased the level of KRP phosphorylation without a subsequent change in contractility. Although phorbol ester does not induce contraction of phasic tissues, the level of KRP phosphorylation is increased. Phosphopeptide maps of KRP from both tissues revealed multiple sites of phosphorylation within the N-terminal region of KRP. Phosphopeptide maps of KRP from gizzard were more complex than those for KRP from artery consistent with heterogeneity at the amino terminus and/or additional sites. We discovered through analysis of KRP phosphorylation in vitro that Ser12, Ser15 and Ser15 are phosphorylated by cAMP-dependent protein kinase, mitogen-activated protein (MAP) kinase and glycogen synthase kinase 3 (GSK3), respectively. Phosphorylation by GSK3 was dependent upon prephosphorylation by MAP kinase. This appears to be the first report of conditional or hierarchical phosphorylation of KRP. Peptides consistent with such multiple phosphorylations were found on the in vivo phosphopeptide maps of avian KRP. Collectively, the available data indicate that there is a complex relationship between the in vivo phosphorylation states of KRP and its effects on relaxation in smooth muscle.

Unique sequence of a high molecular weight myosin light chain kinase is involved in interaction with actin cytoskeleton.

Myosin light chain kinase (MLCK) is the key regulator of cell motility and smooth muscle contraction in higher vertebrates. We searched for the features of the high molecular weight MLCK (MLCK-210) associated with its unique N-terminal sequence not found in a more ubiquitous lower molecular weight MLCK (MLCK-108). MLCK-210 demonstrates stronger association with the Triton-insoluble cytoskeletons than MLCK-108, suggesting the role for this sequence in subcellular targeting. Indeed, the expressed unique domain of MLCK-210 binds and bundles F-actin in vitro and colocalises with the microfilaments in transfected cells reproducing endogenous MLCK-210 distribution. Thus, MLCK-210 features an extensive actin binding interface and, perhaps, acts as an actin cytoskeleton stabiliser.

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.

Screening in a cell-based assay for inhibitors of microglial nitric oxide production reveals calmodulin-regulated protein kinases as potential drug discovery targets.

A high-throughput screening (HTS) assay for inhibitors of nitric oxide (NO) production by activated microglia was developed and used to compare the relative activities of various anti-inflammatory compounds and cell-permeable protein kinase inhibitors. BV-2 cells, an immortalized line that retains phenotypic features of microglia and produces NO in response to lipopolysaccharide (LPS), were used in the activation paradigm for the HTS assay. A characteristic feature of the compounds that were the most potent dose-dependent inhibitors of NO production is their ability to modulate serine/threonine protein kinases. The anti-inflammatory compound K252a, an inhibitor of calmodulin (CaM)-regulated protein kinases, had one of the highest potencies in the assay. Other classes of kinase inhibitors, including the protein kinase A inhibitor H-89, the mitogen activated protein kinase inhibitors PD98059 and SB203580, and the tyrosine kinase inhibitor genistein, were less potent and efficacious than K252a or the general serine/threonine/tyrosine kinase inhibitor staurosporine. K252a suppresses production of the inducible nitric-oxide synthase (iNOS). The inhibitory effect of K252a is not due to cell toxicity and does not correlate with inhibition of NFkappaB nuclear translocation. The mechanism of action appears to involve inhibition of phosphorylation of the transcription factor CREB, a protein whose activity is modulated by phosphorylation by CaM-dependent protein kinases. These data suggest that signal transduction pathways mediated by CaM-dependent protein kinases warrant future study as potential drug discovery targets.

Identification of calcium binding sites in the trypanosome flagellar calcium-acyl switch protein.

The 24 kDa flagellar calcium binding protein (FCaBP) of the protozoan Trypanosoma cruzi is a calcium-acyl switch protein. FCaBP is modified by the addition of myristate and palmitate at its amino terminal segment and both modifications are required for calcium-modulated flagellar membrane association. FCaBP has four sequence motifs for potential calcium binding, and comparison to other calcium-acyl switch proteins, such as recoverin, suggested that only two of these sites are functional. Because it is not possible to predict with certainty the calcium binding affinity or selectivity based on motif analysis alone, we determined the quantitative calcium binding activity of FCaBP by direct ligand binding using the flow dialysis method. The results demonstrated the presence of two calcium binding sites in the full length FCaBP and in a mutant (FCaBPdelta12) lacking the amino terminal pair of sites. FCaBPdelta12 retains its ability to localize to the flagellum. A mutant FCaBP lacking the two carboxyl-terminal sites (FCaBPdelta34), did not bind calcium with high affinity and selectivity under the conditions used. The calcium binding properties of FCaBP are therefore distinct from other myristoyl switch proteins such as recoverin. The results add to a growing body of knowledge about the correlation of sequence motifs with calcium binding activity. Moreover, they demonstrate the need to determine the apparently novel mechanism by which FCaBP undergoes calcium modulated flagellar membrane association and its relation to calcium signal transduction.

Mutation of Lys-75 affects calmodulin conformation.

Some properties of synthetic calmodulin and its five mutants with replacement of Lys-75 were analyzed by means of electrophoresis, limited proteolysis and MALDI mass-spectrometry. A double mutant of calmodulin containing insert KGK between residues 80 and 81 and replacement of Lys-75 by Pro has a highly flexible central helix which is susceptible to trypsinolysis in the presence of Ca2+. Two mutants, K75P and K75E, having a distorted central helix demonstrate high resistance to trypsinolysis in the absence of Ca2+. Arg-90 and Arg-106 being the primary site of trypsinolysis of synthetic calmodulin are partially-protected in K75P and K75E mutants. The central helix of K75A and K75V mutants is stabilized by hydrophobic interactions between residues located in positions 71, 72 and 75. In the presence of Ca2+, the central helix of K75V is resistant to trypsinolysis. Mutations K75A and K75V decrease the rate of trypsinolysis of the central helix with a simultaneous increase of the rate of trypsinolysis in the C-terminal domain of calmodulin. It is concluded that the point mutation in the central helix has a long distance effect on the structure of calmodulin.

Structure-based discovery and in-parallel optimization of novel competitive inhibitors of thymidylate synthase.

BACKGROUND: The substrate sites of enzymes are attractive targets for structure-based inhibitor design. Two difficulties hinder efforts to discover and elaborate new (nonsubstrate-like) inhibitors for these sites. First, novel inhibitors often bind at nonsubstrate sites. Second, a novel scaffold introduces chemistry that is frequently unfamiliar, making synthetic elaboration challenging. RESULTS: In an effort to discover and elaborate a novel scaffold for a substrate site, we combined structure-based screening with in-parallel synthetic elaboration. These techniques were used to find new inhibitors that bound to the folate site of Lactobacillus casei thymidylate synthase (LcTS), an enzyme that is a potential target for proliferative diseases, and is highly studied. The available chemicals directory was screened, using a molecular-docking computer program, for molecules that complemented the three-dimensional structure of this site. Five high-ranking compounds were selected for testing. Activity and docking studies led to a derivative of one of these, dansyltyrosine (Ki 65 microM). Using solid-phase in-parallel techniques 33 derivatives of this lead were synthesized and tested. These analogs are dissimilar to the substrate but bind competitively with it. The most active analog had a Ki of 1.3 microM. The tighter binding inhibitors were also the most specific for LcTS versus related enzymes. CONCLUSIONS: TS can recognize inhibitors that are dissimilar to, but that bind competitively with, the folate substrate. Combining structure-based discovery with in-parallel synthetic techniques allowed the rapid elaboration of this series of compounds. More automated versions of this approach can be envisaged.

Analysis of the functional coupling between calmodulin's calcium binding and peptide recognition properties.

The enhancement of calmodulin's (CaM) calcium binding activity by an enzyme or a recognition site peptide and its diminution by key point mutations at the protein recognition interface (e.g., E84K-CaM), which is more than 20 A away from the nearest calcium ligation structure, can be described by an expanded version of the Adair-Klotz equation for multiligand binding. The expanded equation can accurately describe the calcium binding events and their variable linkage to protein recognition events can be extended to other CaM-regulated enzymes and can potentially be applied to a diverse array of ligand binding systems with allosteric regulation of ligand binding, whether by other ligands or protein interaction. The 1.9 A resolution X-ray crystallographic structure of the complex between E84K-CaM and RS20 peptide, the CaM recognition site peptide from vertebrate smooth muscle and nonmuscle forms of myosin light chain kinase, provides insight into the structural basis of the functional communication between CaM's calcium ligation structures and protein recognition surfaces. The structure reveals that the complex adapts to the effect of the functional mutation by discrete adjustments in the helix that contains E84. This helix is on the amino-terminal side of the helix-loop-helix structural motif that is the first to be occupied in CaM's calcium binding mechanism. The results reported here are consistent with a sequential and cooperative model of CaM's calcium binding activity in which the two globular and flexible central helix domains are functionally linked, and provide insight into how CaM's calcium binding activity and peptide recognition properties are functionally coupled.

Identification of novel classes of protein kinase inhibitors using combinatorial peptide chemistry based on functional genomics knowledge.

A discovery approach based on an intramolecular inhibitory mechanism was applied to a prototype calmodulin (CaM)-regulated protein kinase in order to demonstrate a proof-of-principle for the development of selective inhibitors. The overall approach used functional genomics analysis of myosin light chain kinase (MLCK) to identify short autoinhibitory sequences that lack CaM recognition activity, followed by recursive combinatorial peptide library production and comparative activity screens. Peptide 18 (Arg-Lys-Lys-Tyr-Lys-Tyr-Arg-Arg-Lys-NH2), one of several selective inhibitors discovered, has an IC50 = 50 nM for MLCK, inhibits CaM kinase II only at 4000-fold higher concentrations, and does not inhibit cyclic AMP-dependent protein kinase. Analogues of peptide 18 containing conformationally constrained cis-4-aminocyclohexanecarboxylic acid retained affinity and selectivity. The inhibitors add to the armamentarium available for the deconvolution of complex signal transduction pathways and their relationship to homeostasis and disease, and the approach is potentially applicable to enzymes in which the catalytic and regulatory domains are found within the same open reading frame of a cDNA.

Organization of the genetic locus for chicken myosin light chain kinase is complex: multiple proteins are encoded and exhibit differential expression and localization.

We report that the genetic locus that encodes vertebrate smooth muscle and nonmuscle myosin light chain kinase (MLCK) and kinase-related protein (KRP) has a complex arrangement and a complex pattern of expression. Three proteins are encoded by 31 exons that have only one variation, that of the first exon of KRP, and the genomic locus spans approximately 100 kb of DNA. The three proteins can differ in their relative abundance and localization among tissues and with development. MLCK is a calmodulin (CaM) regulated protein kinase that phosphorylates the light chain of myosin II. The chicken has two MLCK isoforms encoded by the MLCK/KRP locus. KRP does not bind CaM and is not a protein kinase. However, KRP binds to and regulates the structure of myosin II. Thus, KRP and MLCK have the same subcellular target, the myosin II molecular motor system. We examined the tissue and cellular localization of KRP and MLCK in the chicken embryo and in adult chicken tissues. We report on the selective localization of KRP and MLCK among and within tissues and on a differential distribution of the proteins between embryonic and adult tissues. The results fill a void in our knowledge about the organization of the MLCK/KRP genetic locus, which appears to be a late evolving regulatory paradigm, and suggest an independent and complex regulation of expression of the gene products from the MLCK/KRP genetic locus that may reflect a basic principle found in other eukaryotic gene clusters that encode functionally linked proteins.

Sites of interaction between kinase-related protein and smooth muscle myosin.

Kinase-related protein, also known as KRP or telokin, is an independently expressed protein product derived from a gene within the gene for myosin light chain kinase (MLCK). KRP binds to unphosphorylated smooth muscle myosin filaments and stabilizes them against ATP-induced depolymerization in vitro. KRP competes with MLCK for binding to myosin, suggesting that both proteins bind to myosin by the KRP domain (Shirinsky, V. P., Vorotnikov, A. V., Birukov, K. G., Nanaev, A. K., Collinge, M., Lukas, T. J., Sellers, J. R., and Watterson, D. M. (1993) J. Biol. Chem. 268, 16578-16583). In this study, we investigated which regions of myosin and KRP interact in vitro. Using cosedimentation assays, we determined that KRP binds to unphosphorylated myosin with a stoichiometry of 1 mol of KRP/1 mol of myosin and an affinity of 5.5 microM. KRP slows the rate of proteolytic cleavage of the head-tail junction of heavy meromyosin by papain and chymotrypsin, suggesting it is binding to this region of myosin. In addition, competition experiments, using soluble headless fragments of nonmuscle myosin, confirmed that KRP interacts with the regulatory light chain binding region of myosin. The regions important for KRP's binding to myosin were investigated using bacterially expressed KRP truncation mutants. We determined that the acid-rich sequence between Gly138 and Asp151 of KRP is required for high affinity myosin binding, and that the amino terminus and beta-barrel regions weakly interact with myosin. All KRP truncations, at concentrations comparable to their KD values, exhibited some stabilization of myosin filaments against ATP depolymerization in vitro, suggesting that KRP's ability to stabilize myosin filaments is commensurate with its myosin binding affinity. KRP weakened the Km but not the Vmax of phosphorylation of myosin by MLCK, demonstrating that bound KRP does not prevent MLCK from activating myosin.