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J T Stull

Publications and source records attributed to J T Stull.

At least 19 recordsLinked to original sources
PubMed1999

Dynamic modulation of the regulatory domain of myosin heads by pH, ionic strength, and RLC phosphorylation in synthetic myosin filaments.

The position of the myosin head with respect to the filament backbone is thought to be a function of pH, ionic strength (micro) and the extent of regulatory light chain (RLC) phosphorylation [Harrington (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5066-5070]. The object of this study is to examine the dynamics of the proximal part of the myosin head (regulatory domain) which accompany the changes in head disposition. The essential light chain was labeled at Cys177 with the indanedione spin-label followed by the exchange of the labeled proteins into myosin. The mobility of the labeled domain was investigated with saturation transfer electron paramagnetic resonance in reconstituted, synthetic myosin filaments. We have found that the release of the heads from the myosin filament surface by reduction of electrostatic charge is accompanied by a 2-fold increase in the mobility of the regulatory domain. Phosphorylation of the RLC by myosin light chain kinase resulted in a smaller 1. 5-fold increase of motion, establishing that the head disordering observed by electron microscopy [Levine et al. (1996) Biophys. J. 71, 898-907] is due to increased mobility of the heads. This result indirectly supports the hypothesis that the RLC phosphorylation effect on potentiation of force arises from a release of heads from the filament surface and a shift of the heads toward actin.

Animals↗
PubMed1999

Properties of filament-bound myosin light chain kinase.

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.

Actin Cytoskeleton↗
PubMed1998

Calmodulin binding to myosin light chain kinase begins at substoichiometric Ca2+ concentrations: a small-angle scattering study of binding and conformational transitions.

We have used small-angle scattering to study the calcium dependence of the interactions between calmodulin (CaM) and skeletal muscle myosin light chain kinase (MLCK), as well as the conformations of the complexes that form. Scattering data were measured from equimolar mixtures of a functional MLCK and CaM or a mutated CaM (B12QCaM) incompetent to bind Ca2+ in its N-terminal domain, with increasing Ca2+ concentrations. To evaluate differences between CaM-enzyme versus CaM-peptide interactions, similar Ca2+ titration experiments were performed using synthetic peptides based on the CaM-binding sequence from MLCK (MLCK-I). Our data show there are different determinants for CaM binding the isolated peptide sequence compared to CaM binding to the same sequences within the enzyme. For example, binding of either CaM or B12QCaM to the MLCK-I peptide is observed even in the presence of EGTA, whereas binding of CaM to the enzyme requires Ca2+. The peptide studies also show that the conformational collapse of CaM requires both the N and C domains of CaM to be competent for Ca2+ binding as well as interactions with each end of MLCK-I, and it occurs at approximately 2 mol of Ca2+/mol of CaM. We show that CaM binding to the MLCK enzyme begins at substoichiometric concentrations of Ca2+ (< or = 2 mol of Ca2+/mol of CaM), but that the final compact structure of CaM with the enzyme requires saturating Ca2+. In addition, MLCK enzyme does bind to 2Ca2+ x B12QCaM, although this complex is more extended than the complex with native CaM. Our results support the hypothesis that CaM regulation of MLCK involves an initial binding step at less than saturating Ca2+ concentrations and a subsequent activation step at higher Ca2+ concentrations.

Amino Acid Sequence↗
PubMed1998

Impaired metabolic modulation of alpha-adrenergic vasoconstriction in dystrophin-deficient skeletal muscle.

The neuronal isoform of nitric oxide synthase (nNOS) is highly expressed in mammalian skeletal muscle, but its functional role has not been defined. NO has been implicated in the local metabolic regulation of blood flow in contracting skeletal muscle in part by antagonizing sympathetic vasoconstriction. We therefore hypothesized that nNOS in skeletal muscle is the source of the NO mediating the inhibition of sympathetic vasoconstriction in contracting muscle. In the mdx mouse, a model of Duchenne muscular dystrophy in which dystrophin deficiency results in greatly reduced expression of nNOS in skeletal muscle, we found that the normal ability of skeletal muscle contraction to attenuate alpha-adrenergic vasoconstriction is defective. Similar results were obtained in mutant mice that lack the gene encoding nNOS. Together these data suggest a specific role for nNOS in the local metabolic inhibition of alpha-adrenergic vasoconstriction in active skeletal muscle.

Animals↗
PubMed1998

Calmodulin-dependent regulation of inducible and neuronal nitric-oxide synthase.

Neuronal and endothelial nitric-oxide synthases depend upon Ca2+/calmodulin for activation, whereas the activity of the inducible nitric-oxide synthase is Ca2+-independent, presumably due to tightly bound calmodulin. To study these different mechanisms, a series of chimeras derived from neuronal and inducible nitric- oxide synthases were analyzed. Chimeras containing only the oxygenase domain, calmodulin-binding region, or reductase domain of inducible nitric-oxide synthase did not confer significant Ca2+-independent activity. However, each chimera was more sensitive to Ca2+ than the neuronal isoform. The calmodulin-binding region of inducible nitric-oxide synthase with either its oxygenase or reductase domains resulted in significant, but not total, Ca2+-independent activity. Co-immunoprecipitation experiments showed no calmodulin associated with the former chimera in the absence of Ca2+. Trifluoperazine also inhibited this chimera in the absence of Ca2+. The combined interactions of calmodulin bound to inducible nitric-oxide synthase calmodulin-binding region with the oxygenase domain may be weaker than with the reductase domain. Thus, Ca2+-independent activity of inducible nitric-oxide synthase appears to result from the concerted interactions of calmodulin with both the oxygenase and reductase domains in addition to the canonical calmodulin-binding region. The neuronal isoform is not regulated by a unique autoinhibitory element in its reductase domain.

Binding Sites↗
PubMed1998

Neutron-scattering studies reveal further details of the Ca2+/calmodulin-dependent activation mechanism of myosin light chain kinase.

Previously, we utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first low-resolution structure of 4Ca2+.calmodulin (CaM) complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). These experiments showed that, upon binding to MLCK, CaM undergoes a conformational collapse identical to that observed when CaM binds to the isolated peptide corresponding to the CaM binding sequence of MLCK. CaM thereby was shown to release the inhibition of the kinase by inducing a significant movement of its CaM binding and autoinhibitory sequences away from the surface of the catalytic core [Krueger, J. K., Olah, G. A., Rokop, S. E., Zhi, G., Stull, J. T., and Trewhella, J. (1997) Biochemistry 36, 6017-6023]. We report here similar scattering experiments on the CaM.MLCK complex with the addition of substrates; a nonhydrolyzable analogue of adenosine-triphosphate, AMPPNP, and a peptide substrate for MLCK, a phosphorylation sequence from myosin regulatory light chain (pRLC). These substrates are shown to induce an overall compaction of the complex. The separation of the centers-of-mass of the CaM and MLCK components is shortened (by approximately 12 A), thus bringing CaM closer to the catalytic site compared to the complex without substrates. In addition, there appears to be a reorientation of CaM with respect to the kinase upon substrate binding that results in interactions between the N-terminal sequence of CaM and the kinase that were not observed in the complex without substrates. Finally, the kinase itself becomes more compact in the CaM.MLCK.pRLC.AMPPNP complex compared to the complex without substrates. This observed compaction of MLCK upon substrate binding is similar to that arising from the closure of the catalytic cleft in cAMP-dependent protein kinase upon binding pseudosubstrate.

Adenylyl Imidodiphosphate↗
PubMed1998

Skeletal muscle contractions stimulate cGMP formation and attenuate vascular smooth muscle myosin phosphorylation via nitric oxide.

Nitric oxide generated by neuronal nitric oxide synthase in contracting skeletal muscle fibers may regulate vascular relaxation via a cGMP-mediated pathway. Neuronal nitric oxide synthase content is greatly reduced in skeletal muscles from mdx mice. cGMP formation increased in contracting extensor digitorum longus muscles in vitro from C57 control, but not mdx mice. The increase in cGMP content was abolished with NG-nitro-L-arginine. Sodium nitroprusside treatment increased cGMP levels in muscles from both C57 and mdx mice. Skeletal muscle contractions also inhibited phenylephrine-induced phosphorylation of smooth muscle myosin regulatory light chain. Arteriolar dilation was attenuated in contracting muscles from mdx but not C57 mice. NO generated in contracting skeletal muscle may contribute to vasodilation in response to exercise.

Animals↗
PubMed1998

Regulatory segments of Ca2+/calmodulin-dependent protein kinases.

Catalytic cores of skeletal and smooth muscle myosin light chain kinases and Ca2+/calmodulin-dependent protein kinase II are regulated intrasterically by different regulatory segments containing autoinhibitory and calmodulin-binding sequences. The functional properties of these regulatory segments were examined in chimeric kinases containing either the catalytic core of skeletal muscle myosin light chain kinase or Ca2+/calmodulin-dependent protein kinase II with different regulatory segments. Recognition of protein substrates by the catalytic core of skeletal muscle myosin light chain kinase was altered with the regulatory segment of protein kinase II but not with smooth muscle myosin light chain kinase. Similarly, the catalytic properties of the protein kinase II were altered with regulatory segments from either myosin light chain kinase. All chimeric kinases were dependent on Ca2+/calmodulin for activity. The apparent Ca2+/calmodulin activation constant was similarly low with all chimeras containing the skeletal muscle catalytic core. The activation constant was greater with chimeric kinases containing the catalytic core of Ca2+/calmodulin-dependent protein kinase II with its endogenous or myosin light chain kinase regulatory segments. Thus, heterologous regulatory segments affect substrate recognition and kinase activity. Furthermore, the sensitivity to calmodulin activation is determined primarily by the respective catalytic cores, not the calmodulin-binding sequences.

Amino Acid Sequence↗
PubMed1998

Changes in interfilament spacing mimic the effects of myosin regulatory light chain phosphorylation in rabbit psoas fibers.

The modulatory effect of myosin regulatory light chain phosphorylation in mammalian skeletal muscle, first documented as posttetanic potentiation of twitch tension, was subsequently shown to enhance the expression and development of tension at submaximal levels of activating calcium. Structural analyses demonstrated that thick filaments with phosphorylated myosin regulatory light chains appeared disordered: they lost the near-helical, periodic arrangement of myosin head characteristic of the relaxed state. We suggested that disordered heads may be more mobile than ordered heads and are likely to spend more time close to their binding sites on thin filaments. In this study we determined that the physiological effects of phosphorylation could be mimicked by decreasing the lattice spacing between the thick and the thin filaments, either by osmotic compression with dextran or by increasing the sarcomere length of permeabilized rabbit psoas fibers. Phosphorylation of regulatory light chains by incubation of permeabilized fibers with myosin light chain kinase and calmodulin, followed by low levels of activating calcium, potentiated tension development at resting or lower sarcomere lengths in the absence of dextran but had no additional effect on tension potentiation or development in fibers with decreased lattice spacing due to either osmotic compression or increased sarcomere length.

Animals↗
PubMed1998

Structural and functional responses of mammalian thick filaments to alterations in myosin regulatory light chains.

The ordered array of myosin heads, characteristic of relaxed striated muscle thick filaments, is reversibly disordered by phosphorylating myosin regulatory light chains, decreasing temperature and/or ionic strength, increasing pH, and depleting nucleotide. In the case of light chain phosphorylation, disorder, most likely due to a change in charge affecting the light chain amino-terminus, reflects increased myosin head mobility, thus increased accessibility to actin, and results in increased calcium sensitivity of tension development. Thus, interactions between the unphosphorylated regulatory light chain and the filament backbone may help maintain the overall order of the relaxed filament. To define this relationship, we have examined the structural and functional effects of such manipulations as exchanging wild-type smooth and skeletal myosin light chains into permeabilized rabbit psoas fibers and removing regulatory light chains (without exchange) from such fibers. We have also compared the structural and functional parameters of biopsied fibers from patients with severe familial hypertrophic cardiomyopathy due to a single amino acid substitution in the regulatory light chains to those exhibited by fibers from normal relatives. Our results support a role for regulatory light chains in reversible ordering of myosin heads and suggest that economy of energy utilization may provide for evolutionary preservation of this function in vertebrate striated muscle.

Actin Cytoskeleton↗
PubMed1998

Sparing of mdx extraocular muscles from dystrophic pathology is not attributable to normalized concentration or distribution of neuronal nitric oxide synthase.

Previous findings have led to speculations that decreased concentration of nNOS (neuronal nitric oxide synthase) may underlie some aspects of the pathophysiology of dystrophic muscle. We have tested whether the sparing of extraocular muscles (EOM) in muscular dystrophy is attributable to the presence of normal nNOS concentration and distribution in these muscles. Measurements of total nNOS concentration in control muscle showed that total nNOS comprises approximately 0.05% of total muscle protein, indicating a molar stoichiometry of approximately 60 and 20 to total dystrophin and syntrophin, respectively. Thus, most muscle nNOS is either not associated with the dystrophin complex, or binds to yet unidentified sites in the complex. nNOS concentration was at least two-fold greater in C57 EOM and tibialis anterior (TA) compared with mdx samples. No significant differences in nNOS concentration in EOM versus TA in either mdx or C57 mice were observed, nNOS was concentrated at the sarcolemma of all C57 samples, while mdx nNOS displayed a cytosolic distribution, except in fibers that reverted to express dystrophin. These data show that mdx EOM are spared by a mechanism other than normalized concentration and location of nNOS.

Aging↗
PubMed1998

Myosin light chain kinase: functional domains and structural motifs.

Conventional myosin light chain kinase found in differentiated smooth and non-muscle cells is a dedicated Ca2+/calmodulin-dependent protein kinase which phosphorylates the regulatory light chain of myosin II. This phosphorylation increases the actin-activated myosin ATPase activity and is thought to play major roles in a number of biological processes, including smooth muscle contraction. The catalytic domain contains residues on its surface that bind a regulatory segment resulting in autoinhibition through an intrasteric mechanism. When Ca2+/calmodulin binds, there is a marked displacement of the regulatory segment from the catalytic cleft allowing phosphorylation of myosin regulatory light chain. Kinase activity depends upon Ca2+/calmodulin binding not only to the canonical calmodulin-binding sequence but also to additional interactions between Ca2+/calmodulin and the catalytic core. Previous biochemical evidence shows myosin light chain kinase binds tightly to actomyosin containing filaments. The kinase has low-affinity myosin and actin binding sites in Ig-like motifs at the N- and C-terminus, respectively. Recent results show the N-terminus of myosin light chain kinase is responsible for filament binding in vivo. However, the apparent binding affinity is greater for smooth muscle myofilaments, purified thin filaments, or actin-containing filaments in permeable cells than for purified smooth muscle F-actin or actomyosin filaments from skeletal muscle. These results suggest a protein on actin thin filaments that may facilitate kinase binding. Myosin light chain kinase does not dissociate from filaments in the presence of Ca2+/calmodulin raising the interesting question as to how the kinase phosphorylates myosin in thick filaments if it is bound to actin-containing thin filaments.

Animals↗
PubMed1998

Mechanical loading regulates NOS expression and activity in developing and adult skeletal muscle.

The hypothesis that changes in muscle activation and loading regulate the expression and activity of neuronal nitric oxide (NO) synthase (nNOS) was tested using in vitro and in vivo approaches. Removal of weight bearing from rat hindlimb muscles for 10 days resulted in a significant decrease in nNOS protein and mRNA concentration in soleus muscles, which returned to control concentrations after return to weight bearing. Similarly, the concentration of nNOS in cultured myotubes increased by application of cyclic loading for 2 days. NO release from excised soleus muscles was increased significantly by a single passive stretch of 20% or by submaximal activation at 2 Hz, although the increases were not additive when both stimuli were applied simultaneously. Increased NO release resulting from passive stretch or activation was dependent on the presence of extracellular calcium. Cyclic loading of cultured myotubes also resulted in a significant increase in NO release. Together, these findings show that activity of muscle influences NO production in the short term, by regulating NOS activity, and in the long term, by regulating nNOS expression.

Aging↗
PubMed1997

Structures of calmodulin and a functional myosin light chain kinase in the activated complex: a neutron scattering study.

Calmodulin (CaM) is the major intracellular receptor for Ca2+ and is responsible for the Ca2+-dependent regulation of a wide variety of cellular processes via interactions with a diverse array of target enzymes. Our current view of the structural basis for CaM enzyme activation is based on biophysical studies of CaM complexed with small peptides that model CaM-binding domains. A major concern with interpreting data from these structures in terms of target enzyme activation mechanisms is that the larger enzyme structure might be expected to impose constraints on CaM binding. Full understanding of the molecular mechanism for CaM-dependent enzyme activation requires additional structural information on the interaction of CaM with functional enzymes. We have utilized small-angle X-ray scattering and neutron scattering with contrast variation to obtain the first structural view of CaM complexed with a functional enzyme, an enzymatically active truncation mutant of skeletal muscle myosin light chain kinase (MLCK). Our data show that CaM undergoes an unhindered conformational collapse upon binding MLCK and activates the enzyme by inducing a significant movement of the kinase's CaM binding and autoinhibitory sequences away from the surface of the catalytic core.

Calmodulin↗
PubMed1997

Binding of myosin light chain kinase to cellular actin-myosin filaments.

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.

Actin Cytoskeleton↗
PubMed1997

Localization of an actin binding domain in smooth muscle myosin light chain kinase.

Phosphorylation of the regulatory light chain of myosin II by myosin light chain kinase is important for regulating many contractile processes. Smooth muscle myosin light chain kinase has been shown to be associated with both actin and myosin filaments in vitro and in vivo. In this report we define an actin binding region by using molecular deletions to generate recombinant mutant proteins that were analyzed by co-sedimentation with F-actin. An actin binding region restricted to residues 2-42 in the amino terminus of the rabbit smooth muscle myosin light chain kinase was identified.

Actins↗