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James T. Stull - One of the best experts on this subject based on the ideXlab platform.
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myosin Light Chain kinase and the role of myosin Light Chain phosphorylation in skeletal muscle
Archives of Biochemistry and Biophysics, 2011Co-Authors: James T. Stull, Kristine E Kamm, Rene VandenboomAbstract:Abstract Skeletal muscle myosin Light Chain kinase (skMLCK) is a dedicated Ca 2+ /calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory Light Chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca 2+ binding to calmodulin forming a (Ca 2+ ) 4 •calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca 2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca 2+ /calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin Light Chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca 2+ -sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.
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signaling to myosin regulatory Light Chain in sarcomeres
Journal of Biological Chemistry, 2011Co-Authors: Kristine E Kamm, James T. StullAbstract:Myosin regulatory Light Chain (RLC) phosphorylation in skeletal and cardiac muscles modulates Ca2+-dependent troponin regulation of contraction. RLC is phosphorylated by a dedicated Ca2+-dependent myosin Light Chain kinase in fast skeletal muscle, where biochemical properties of RLC kinase and phosphatase converge to provide a biochemical memory for RLC phosphorylation and post-activation potentiation of force development. The recent identification of cardiac-specific myosin Light Chain kinase necessary for basal RLC phosphorylation and another potential RLC kinase (zipper-interacting protein kinase) provides opportunities for new approaches to study signaling pathways related to the physiological function of RLC phosphorylation and its importance in cardiac muscle disease.
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Properties of filament-bound myosin Light Chain kinase.
Journal of Biological Chemistry, 1999Co-Authors: Katherine Luby-phelps, James T. StullAbstract:Abstract Myosin Light Chain kinase binds to actin-containing filaments from cells with a greater affinity than to F-actin. However, it is not known if this binding in cells is regulated by Ca2+/calmodulin as it is with F-actin. Therefore, the binding properties of the kinase to stress fibers were examined in smooth muscle-derived A7r5 cells. Full-length myosin Light Chain kinase or a truncation mutant lacking residues 2–142 was expressed as chimeras containing green fluorescent protein at the C terminus. In intact cells, the full-length kinase bound to stress fibers, whereas the truncated kinase showed diffuse fluorescence in the cytoplasm. After permeabilization with saponin, the fluorescence from the truncated kinase disappeared, whereas the fluorescence of the full-length kinase was retained on stress fibers. Measurements of fluorescence intensities and fluorescence recovery after photobleaching of the full-length myosin Light Chain kinase in saponin-permeable cells showed that Ca2+/calmodulin did not dissociate the kinase from these filaments. However, the filament-bound kinase was sufficient for Ca2+-dependent phosphorylation of myosin regulatory Light Chain and contraction of stress fibers. Thus, dissociation of myosin Light Chain kinase from actin-containing thin filaments is not necessary for phosphorylation of myosin Light Chain in thick filaments. We note that the distance between the N terminus and the catalytic core of the kinase is sufficient to span the distance between thin and thick filaments.
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Binding of Myosin Light Chain Kinase to Cellular Actin-Myosin Filaments
Journal of Biological Chemistry, 1997Co-Authors: Katherine Luby-phelps, James T. StullAbstract:Abstract Myosin Light Chain kinase binds to the actomyosin-containing filaments in smooth and nonmuscle cells. However, the region of the kinase necessary for this high affinity binding in vivo is not known, although it has been proposed that the N and C termini bind to actin and myosin in vitro, respectively. Truncated myosin Light Chain kinases containing the catalytic core and calmodulin-binding domain but lacking N (amino acids 1-655) and/or C (amino acids 1004-1147) termini were expressed in the baculovirus system and purified. All enzymes were catalytically active and Ca2+/calmodulin-dependent. The C-terminal truncated myosin Light Chain kinase bound to detergent-washed smooth muscle contractile proteins similar to recombinant full-length myosin Light Chain kinase or enzyme purified from smooth muscle. The apparent affinity of the full-length kinase was greater for the actomyosin-containing filaments with associated proteins than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. In contrast, truncations at the N terminus alone or at both N and C termini resulted in no significant binding. Similar effects were observed by two other assays: binding of fluorescently labeled myosin Light Chain kinases to actin-containing stress fibers in detergent-treated fibroblasts and localization of fluorescently labeled kinases after microinjection into primary smooth muscle cells in culture. The full-length and the C-terminal truncated myosin Light Chain kinases, but not myosin Light Chain kinases truncated at the N terminus or both N and C termini, associated with filaments in cells. Thus, the N terminus and not the C terminus of myosin Light Chain kinase is necessary for high affinity binding to actomyosin-containing filaments in smooth and nonmuscle cells.
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Phosphorylation of myosin Light Chain kinase: a cellular mechanism for Ca2+ desensitization
Molecular and Cellular Biochemistry, 1993Co-Authors: James T. Stull, Da Chun Tang, Malu G Tansey, R. Ann Word, Kristine E KammAbstract:Phosphorylation of the regulatory Light Chain of myosin by the Ca2+/calmodulin-dependent myosin Light Chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin Light Chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca2+/calmodulin-dependent protein kinase II phosphorylates myosin Light Chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca2+/calmodulin required for activation and hence increases the Ca2+ concentrations required for myosin Light Chain kinase activity in cells. However, the concentration of cytosolic Ca2+ required to effect myosin Light Chain kinase phosphorylation is greater than that required for myosin Light Chain phosphorylation. Phosphorylation of myosin Light Chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca2+ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin Light Chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin Light Chain phosphorylation.
Katalin Ajtai - One of the best experts on this subject based on the ideXlab platform.
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Light Chain Kinase Specificity in Cardiac Myosin
Biophysical Journal, 2012Co-Authors: Matthew P. Josephson, Laura A. Sikkink, Alan R. Penheiter, Thomas P. Burghardt, Katalin AjtaiAbstract:Human ventricular cardiac myosin regulatory Light Chain (MYL2) phosphorylation modifies Ser15. This modification affects MYL2 secondary structure and modulates the Ca2+ sensitivity of contraction in cardiac tissue. Smooth muscle myosin Light Chain kinase (smMLCK) is prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated Ser15 in MYL2 in vitro. Specific modification of Ser15 was verified by direct detection of the phospho group on Ser15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory Light Chain Ser15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (Ser20 in smooth muscle) but failed to phosphorylate the myosin regulatory Light Chain in rabbit skeletal myosin. Michaelis-Menten Vm and KM constants for Ser15 phosphorylation in MYL2, porcine ventricular myosin, and chicken gizzard myosin are similar. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression. Supported by NIH NIAMS and NHLBI grants R01AR049277 and R01HL095572.
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Smooth Muscle Myosin Light Chain Kinase Efficiently Phosphorylates Serine 15 of Cardiac Myosin Regulatory Light Chain
Biochemical and Biophysical Research Communications, 2011Co-Authors: Matthew P. Josephson, Laura A. Sikkink, Alan R. Penheiter, Thomas P. Burghardt, Katalin AjtaiAbstract:HighLights: Black-Right-Pointing-Pointer Cardiac myosin regulatory Light Chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin Light Chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory Light Chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin Light Chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory Light Chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory Light Chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicatesmore » similar Michaelis-Menten V{sub max} and K{sub M} for regulatory Light Chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.« less
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Smooth muscle myosin Light Chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory Light Chain.
Biochemical and biophysical research communications, 2011Co-Authors: Matthew P. Josephson, Laura A. Sikkink, Alan R. Penheiter, Thomas P. Burghardt, Katalin AjtaiAbstract:Specific phosphorylation of the human ventricular cardiac myosin regulatory Light Chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin Light Chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory Light Chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory Light Chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory Light Chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.
Kristine E Kamm - One of the best experts on this subject based on the ideXlab platform.
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myosin Light Chain kinase and the role of myosin Light Chain phosphorylation in skeletal muscle
Archives of Biochemistry and Biophysics, 2011Co-Authors: James T. Stull, Kristine E Kamm, Rene VandenboomAbstract:Abstract Skeletal muscle myosin Light Chain kinase (skMLCK) is a dedicated Ca 2+ /calmodulin-dependent serine–threonine protein kinase that phosphorylates the regulatory Light Chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca 2+ binding to calmodulin forming a (Ca 2+ ) 4 •calmodulin complex sufficient for activation with a diffusion limited, stoichiometric binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca 2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca 2+ /calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin Light Chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca 2+ -sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue.
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signaling to myosin regulatory Light Chain in sarcomeres
Journal of Biological Chemistry, 2011Co-Authors: Kristine E Kamm, James T. StullAbstract:Myosin regulatory Light Chain (RLC) phosphorylation in skeletal and cardiac muscles modulates Ca2+-dependent troponin regulation of contraction. RLC is phosphorylated by a dedicated Ca2+-dependent myosin Light Chain kinase in fast skeletal muscle, where biochemical properties of RLC kinase and phosphatase converge to provide a biochemical memory for RLC phosphorylation and post-activation potentiation of force development. The recent identification of cardiac-specific myosin Light Chain kinase necessary for basal RLC phosphorylation and another potential RLC kinase (zipper-interacting protein kinase) provides opportunities for new approaches to study signaling pathways related to the physiological function of RLC phosphorylation and its importance in cardiac muscle disease.
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Phosphorylation of myosin Light Chain kinase: a cellular mechanism for Ca2+ desensitization
Molecular and Cellular Biochemistry, 1993Co-Authors: James T. Stull, Da Chun Tang, Malu G Tansey, R. Ann Word, Kristine E KammAbstract:Phosphorylation of the regulatory Light Chain of myosin by the Ca2+/calmodulin-dependent myosin Light Chain kinase plays an important role in smooth muscle contraction, nonmuscle cell shape changes, platelet contraction, secretion, and other cellular processes. Smooth muscle myosin Light Chain kinase is also phosphorylated, and recent results from experiments designed to satisfy the criteria of Krebs and Beavo for establishing the physiological significance of enzyme phosphorylation have provided insights into the cellular regulation and function of this phosphorylation in smooth muscle. The multifunctional Ca2+/calmodulin-dependent protein kinase II phosphorylates myosin Light Chain kinase at a regulatory site near the calmodulin-binding domain. This phosphorylation increases the concentration of Ca2+/calmodulin required for activation and hence increases the Ca2+ concentrations required for myosin Light Chain kinase activity in cells. However, the concentration of cytosolic Ca2+ required to effect myosin Light Chain kinase phosphorylation is greater than that required for myosin Light Chain phosphorylation. Phosphorylation of myosin Light Chain kinase is only one of a number of mechanisms used by the cell to down regulate the Ca2+ signal in smooth muscle. Since both smooth and nonmuscle cells express the same form of myosin Light Chain kinase, this phosphorylation may play a regulatory role in cellular processes that are dependent on myosin Light Chain phosphorylation.
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GTP/ggS-Induced phosphorylation of myosin Light Chain kinase in smooth muscle
FEBS Letters, 1993Co-Authors: Da Chun Tang, Kristine E Kamm, Yasutaka Kubota, James T. StullAbstract:Phosphorylation of myosin Light Chain kinase by a Ca2+-dependent protein kinase increases the concentration of Ca2+/calmodulin required for half-maximal activation. The Ca2+ concentrations required for myosin Light Chain kinase phosphorylation in permeable smooth muscle are similar to those required for myosin Light Chain phosphorylation. Both GTPγS and carbachol increase the Ca2+ sensitivity of myosin Light Chain kinase phosphorylation as well as Light Chain phosphorylation. It is proposed that a similar G-protein mediated mechanism regulates the Ca2+-dependent phosphorylation of these two contractile proteins in smooth muscle.
Matthew P. Josephson - One of the best experts on this subject based on the ideXlab platform.
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Light Chain Kinase Specificity in Cardiac Myosin
Biophysical Journal, 2012Co-Authors: Matthew P. Josephson, Laura A. Sikkink, Alan R. Penheiter, Thomas P. Burghardt, Katalin AjtaiAbstract:Human ventricular cardiac myosin regulatory Light Chain (MYL2) phosphorylation modifies Ser15. This modification affects MYL2 secondary structure and modulates the Ca2+ sensitivity of contraction in cardiac tissue. Smooth muscle myosin Light Chain kinase (smMLCK) is prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated Ser15 in MYL2 in vitro. Specific modification of Ser15 was verified by direct detection of the phospho group on Ser15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory Light Chain Ser15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (Ser20 in smooth muscle) but failed to phosphorylate the myosin regulatory Light Chain in rabbit skeletal myosin. Michaelis-Menten Vm and KM constants for Ser15 phosphorylation in MYL2, porcine ventricular myosin, and chicken gizzard myosin are similar. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression. Supported by NIH NIAMS and NHLBI grants R01AR049277 and R01HL095572.
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Smooth Muscle Myosin Light Chain Kinase Efficiently Phosphorylates Serine 15 of Cardiac Myosin Regulatory Light Chain
Biochemical and Biophysical Research Communications, 2011Co-Authors: Matthew P. Josephson, Laura A. Sikkink, Alan R. Penheiter, Thomas P. Burghardt, Katalin AjtaiAbstract:HighLights: Black-Right-Pointing-Pointer Cardiac myosin regulatory Light Chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin Light Chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory Light Chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin Light Chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory Light Chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory Light Chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicatesmore » similar Michaelis-Menten V{sub max} and K{sub M} for regulatory Light Chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.« less
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Smooth muscle myosin Light Chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory Light Chain.
Biochemical and biophysical research communications, 2011Co-Authors: Matthew P. Josephson, Laura A. Sikkink, Alan R. Penheiter, Thomas P. Burghardt, Katalin AjtaiAbstract:Specific phosphorylation of the human ventricular cardiac myosin regulatory Light Chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca(2+) sensitivity of contraction in cardiac tissue. Smooth muscle myosin Light Chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory Light Chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory Light Chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V(max) and K(M) for regulatory Light Chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.
B. P. Herring - One of the best experts on this subject based on the ideXlab platform.
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Substrate specificity of myosin Light Chain kinases.
Journal of Biological Chemistry, 1992Co-Authors: B. P. Herring, Patricia J. Gallagher, James T. StullAbstract:Abstract Skeletal muscle myosin Light Chain kinase can phosphorylate myosin Light Chains isolated from skeletal or smooth muscle. In contrast, smooth muscle myosin Light Chain kinase specifically phosphorylates Light Chains isolated from smooth muscle. In this study, we have identified residues within the rabbit smooth and skeletal muscle myosin Light Chain kinases which may interact with the basic residues that are important substrate determinants in the Light Chains. Mutation of aspartic acid 270 amino-terminal of the catalytic core of the skeletal muscle myosin Light Chain kinase increased the Km value for both smooth and skeletal muscle Light Chains. Although deletions of the analogous region of the smooth muscle myosin Light Chain kinase (residues 663-678) markedly increased the Km value for Light Chain, mutation of any single acidic residue within this region did not have a similar effect. Mutation of single residues within the catalytic core of the skeletal muscle (E377 and E421) and smooth muscle (E777 and E821) myosin Light Chain kinases increased Km values for the smooth muscle Light Chain at least 35- and 100-fold, respectively. It is proposed that these residues may form ionic interactions with the arginine that is 3 residues amino-terminal of the phosphorylatable serine in the smooth muscle Light Chain.
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Biochemical properties of chimeric skeletal and smooth muscle myosin Light Chain kinases.
Journal of Biological Chemistry, 1992Co-Authors: Sancy A Leachman, B. P. Herring, Patricia J. Gallagher, Michael J. Mcphaul, James T. StullAbstract:Abstract The molecular and biochemical properties of myosin Light Chain kinases from chicken skeletal and smooth muscle were investigated by recombinant DNA techniques. Deletion of the amino-terminal region of either the smooth or skeletal muscle myosin Light Chain kinase resulted in a decrease in Vmax with no significant change in Km values for Light Chain substrates. Skeletal/smooth muscle chimeric kinases were inactive when a 65-residue region amino-terminal of the catalytic core was exchanged between the two forms. Changing alanine 494 to glutamic acid within this region in the chicken skeletal muscle myosin Light Chain kinase increased the Km values for Light Chains 10-fold. These results are consistent with the hypothesis that the region amino-terminal of the catalytic core in myosin Light Chain kinases is involved in Light Chain recognition. A skeletal muscle kinase which contained the smooth muscle calmodulin binding domain remained regulated by Ca2+/calmodulin. Thus, the calmodulin binding domains of smooth and skeletal muscle myosin Light Chain kinases share structural elements necessary for regulation.
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Molecular characterization of a mammalian smooth muscle myosin Light Chain kinase.
Journal of Biological Chemistry, 1991Co-Authors: Patricia J. Gallagher, B. P. Herring, S. A. Griffin, James T. StullAbstract:Abstract A 5.6-kilobase cDNA clone has been isolated which includes the entire coding region for the myosin Light Chain kinase from rabbit uterine tissue. This cDNA, expressed in COS cells, encodes a Ca2+/calmodulin-dependent protein kinase with catalytic properties similar to other purified smooth muscle myosin Light Chain kinases. A module (TLKPVGNIKPAE), repeated sequentially 15 times, has been identified near the N terminus of this smooth muscle kinase. It is not present in chicken gizzard or rabbit skeletal muscle myosin Light Chain kinases. This repeat module and a subrepeat (K P A/V) are similar in amino acid content to repeated motifs present in other proteins, some of which have been shown to associate with chromatin structures. Immunoblot analysis after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, used to compare myosin Light Chain kinase present in rabbit, bovine, and chicken smooth and nonmuscle tissues, showed that within each species both tissue types have myosin Light Chain kinases with indistinguishable molecular masses. These data suggest that myosin Light Chain kinases present in smooth and nonmuscle tissues are the same protein.