Vladimir P. Shirinsky

Stretch affects phenotype and proliferation of vascular smooth muscle cells

The exertion of periodic dynamic strain on the arterial wall is hypothesized to be relevant to smooth muscle cell morphology and function. This study has investigated the effect of cyclic mechanical stretching on rabbit aortic smooth muscle cell proliferation and expression of contractile phenotype protein markers. Cells were cultured on flexible-bottomed dishes and cyclic stretch was applied (frequency 30 cycles/min, 15% elongation) using a Flexercell Strain unit. Cyclic stretch potentiated smooth muscle cell proliferation in serum-activated cultures but not in cultures maintained in 0.5% fetal calf serum. Stretching induced a serum-independent increase of h-caldesmon expression and this effect was reversible following termination of mechanical stimulation. Strain was without effect on smooth muscle myosin or calponin expression. In cells grown on laminin stretch-induced h-caldesmon expression was more prominent than in cells cultured on collagen types I and IV, poly-L-lysine and gelatin. These data suggest that cyclic mechanical stimulation possesses dual effect on vascular smooth muscle cell phenotype characteristics since it: 1) potentiates proliferation, an attribute of a dedifferentiated phenotype; and 2) increases expression of h-caldesmon considered a marker of a differentiated smooth muscle cell state.

Protein kinase involved in lung injury susceptibility: Evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment

Acute lung injury (ALI) associated with sepsis and iatrogenic ventilator-induced lung injury resulting from mechanical ventilation are major medical problems with an unmet need for small molecule therapeutics. Prevailing hypotheses identify endothelial cell (EC) layer dysfunction as a cardinal event in the pathophysiology, with intracellular protein kinases as critical mediators of normal physiology and possible targets for drug discovery. The 210,000 molecular weight myosin light chain kinase (MLCK210, also called EC MLCK because of its abundance in EC) is hypothesized to be important for EC barrier function and might be a potential therapeutic target. To test these hypotheses directly, we made a selective MLCK210 knockout mouse that retains production of MLCK108 (also called smooth-muscle MLCK) from the same gene. The MLCK210 knockout mice are less susceptible to ALI induced by i.p. injection of the endotoxin lipopolysaccharide and show enhanced survival during subsequent mechanical ventilation. Using a complementary chemical biology approach, we developed a new class of small-molecule MLCK inhibitor based on the pharmacologically privileged aminopyridazine and found that a single i.p. injection of the inhibitor protected WT mice against ALI and death from mechanical ventilation complications. These convergent results from two independent approaches demonstrate a pivotal in vivo role for MLCK in susceptibility to lung injury and validate MLCK as a potential drug discovery target for lung injury.

Intraluminal Pressure Is Essential for the Maintenance of Smooth Muscle Caldesmon and Filamin Content in Aortic Organ Culture

Different forms of mechanical stimulation are among the physiological factors constantly acting on the vessel wall. We previously demonstrated that subjecting vascular smooth muscle cells (VSMCs) in culture to cyclic stretch increased the expression of high-molecular-weight caldesmon, a marker protein of a differentiated, contractile, VSMC phenotype. In the present work the effects of mechanical factors, in the form of circumferential stress and shear stress, on the characteristics of SM contractile phenotype were studied in an organ culture of rabbit aorta. Application of an intralumininal pressure of 80 mm Hg to aortic segments cultured in Dulbeccos modified Eagles medium containing 20% fetal calf serum for 3 days prevented the decrease in high-molecular-weight caldesmon content (70+/-4% of initial level in nonpressurized vessel, 116+/-17% at 80 mm Hg) and filamin content (80+/-5% in nonpressurized vessel, 100+/-2% at 80 mm Hg). SM myosin and low-molecular-weight caldesmon contents showed no dependence on vessel pressurization. Neither endothelial denudation nor alteration of intraluminal flow rates affected marker protein content in 3-day vessel culture, thus excluding the possibility of any shear or endothelial effects. Maintenance of high high-molecular-weight caldesmon and filamin levels in the organ cultures of pressurized and stretched vessels demonstrates the positive role of mechanical factors in the control of the VSMC differentiated phenotype.

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 μm. 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 β-barrel regions weakly interact with myosin. All KRP truncations, at concentrations comparable to theirK D 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 K m but not the V max of phosphorylation of myosin by MLCK, demonstrating that bound KRP does not prevent MLCK from activating myosin.

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. J. Cell. Biochem. 70:402–413, 1998.

Multiple gene products are produced from a novel protein kinase transcription region

The nonmuscle/smooth muscle myosin light chain kinase (MLCK) and the kinase related protein (KRP) that lacks protein kinase activity are myosin II binding proteins encoded in the vertebrate genome by a true gene within a gene relationship. The genomic organization and expression result in the same amino acid sequence in different molecular contexts from two different sizes of mRNA. We report here the identification and characterization of a third size class of gene products. The protein appears to be a higher molecular weight form of MLCK with additional amino terminal tail sequence which might provide differential subcellular targeting characteristics.

Expression of calponin in rabbit and human aortic smooth muscle cells

SummaryPolyclonal antibodies to chicken gizzard calponin were used to localize calponin and determine calponin expression in rabbit and human aortic smooth muscle cells in culture. Calponin was localized on the microfilament bundles of cultured smooth muscle cells. Early in primary culture,ccalponin staining was accumulated preferentially in the central part of the cell body. With time in culture, the number of calponin-negative smooth muscle cells increased while the distribution of calponin in calponin-positive cells became more even along the stress fibers. Calponin content and the calponin/actin ratio decreased about 5-fold in rabbit aortic smooth muscle cells during the first week in primary culture and remained low in proliferating cells. The same tendency in calponin expression was observed when human vascular smooth muscle was studied. On cryostat sections of human umbilical cord, calponin antibodies mainly stained vessel walls of both the arteries and veins, although less intensive labelling was also observed in non-vascular tissue. When primary isolates of human aortic intimal and medial smooth muscle cells were compared with corresponding passaged cultures, it was found that calponin content was reduced about 9-fold in these cells in culture and was similar to the amount of calponin in endothelial cells and fibroblasts. Thus, high calponin expression may be used as an additional marker of vascular smooth muscle cell contractile phenotype.

Phosphorylation of smooth muscle caldesmon by three protein kinases: implication for domain mapping

Phosphorylation of duck gizzard caldesmon by Ca2+/phospholipid‐dependent protein kinase, Ca2+/calmodulin‐dependent protein kinase and casein kinase II has been investigated. The Ca2+/phospholipid‐dependent protein kinase incorporates more than 3 mol phosphate per mol (140 kDa) caldesmon. All phosphorylation sites are localized in the actin‐ and calmodulin‐binding peptide (40–45 kDa) supposed to be a part of the C‐terminal domain of caldesmon. Casein kinase II phosphorylates only one site located in a short (25–27 kDa) peptide, presumably in the caldesmon N‐terminal domain. The Ca2+/calmodulin‐dependent protein kinase phosphorylates two sites located in the N‐ and C‐terminal domains of caldesmon.

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. J. Cell. Biochem. 75:481–491, 1999.

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.