Joseph M. Chalovich

Inhibition of contraction and myosin light chain phosphorylation in guinea-pig smooth muscle by p21-activated kinase 1

The p21‐activated protein kinases (PAKs) have been implicated in cytoskeletal rearrangements and modulation of non‐muscle contractility. Little, however, is known about the role of the PAK family members in smooth muscle contraction. Therefore, we investigated the effect of the predominant isoform in vascular smooth muscle cells, PAK1, on contraction and phosphorylation of the regulatory light chains of myosin (r‐MLC) in Triton‐skinned guinea‐pig smooth muscle. We also investigated which of the three putative substrates at the contractile apparatus ‐ MLCK, caldesmon or r‐MLC ‐ is phosphorylated by PAK1 in smooth muscle tissue. Incubation of Triton‐skinned carotid artery and taenia coli from guinea‐pig with an active mutant of PAK1 in relaxing solution for 30–60 min resulted in inhibition of submaximal force by about 50 %. The mechanism of inhibition of force was studied in the Triton‐skinned taenia coli. In this preparation, inhibition of force was associated with a respective inhibition of r‐MLC phosphorylation. In the presence of the myosin phosphatase inhibitor, microcystin‐LR (10 μm), the rate of contraction and r‐MLC phosphorylation elicited at pCa 6.79 were both decreased. Because under these conditions the rate of r‐MLC phosphorylation is solely dependent on MLCK activity, this result suggests that the inhibitory effect of PAK1 on steady‐state force and r‐MLC phosphorylation is due to inhibition of MLCK. In line with this, we found that MLCK was significantly phosphorylated by PAK1 while there was very little 32P incorporation into caldesmon. PAK1 phosphorylated isolated r‐MLC but not those in the skinned fibres or in purified smooth muscle myosin II. In conclusion, these results suggest that PAK1 attenuates contraction of skinned smooth muscle by phosphorylating and inhibiting MLCK.

Conservation of the regulated structure of folded myosin 2 in species separated by at least 600 million years of independent evolution

The myosin 2 family of molecular motors includes isoforms regulated in different ways. Vertebrate smooth-muscle myosin is activated by phosphorylation of the regulatory light chain, whereas scallop striated adductor-muscle myosin is activated by direct calcium binding to its essential light chain. The paired heads of inhibited molecules from myosins regulated by phosphorylation have an asymmetric arrangement with motor–motor interactions. It was unknown whether such interactions were a common motif for inactivation used in other forms of myosin-linked regulation. Using electron microscopy and single-particle image processing, we show that indistinguishable structures are indeed found in myosins and heavy meromyosins isolated from scallop striated adductor muscle and turkey gizzard smooth muscle. The similarities extend beyond the shapes of the heads and interactions between them: In both myosins, the tail folds into three segments, apparently at identical sites; all three segments are in close association outside the head region; and two segments are associated in the same way with one head in the asymmetric arrangement. Thus, these organisms, which have different regulatory mechanisms and diverged from a common ancestor >600 Myr ago, have the same quaternary structure. Conservation across such a large evolutionary distance suggests that this conformation is of fundamental functional importance.

Identification of an unknown 31P nuclear magnetic resonance from dystrophic chicken as l-serine ethanolamine phosphodiester

Abstract A phosphodiester, which comes into resonance at 0.4 ppm in the 31 P nuclear magnetic resonance spectrum of intact muscles, has been isolated from the pectoralis muscle of chickens with hereditary muscular dystrophy by perchloric acid extraction, barium and alcoholic fractionation, and chromatographic isolation procedures. The compound, l -serine ethanolamine phosphodiester, whose presence is a characteristic of the diseased chicken muscle, has been characterized by 31 P, 13 C, and 1 H nuclear magnetic resonance as well as by chemical and chromatographic procedures.

Kinetics of thin filament activation probed by fluorescence of N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-labeled troponin I incorporated into skinned fibers of rabbit psoas muscle: implications for regulation of muscle contraction.

Making use of troponin with fluorescently labeled troponin I subunit (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole-troponin I, IANBD-TnI) that had previously been described in solution studies as a probe for thin filament activation (. Proc. Natl. Acad. Sci. 77:7209-7213), we present a new approach that allows the kinetics of thin filament activation to be studied in skinned muscle fibers. After the exchange of native troponin for fluorescently labeled troponin, the fluorescence intensity is sensitive to both changes in calcium concentration and actin attachment of cross-bridges in their strong binding states (. Biophys. J. 77:000-000). Imposing rapid changes in the fraction of strongly attached cross-bridges, e.g., by switching from isometric contraction to high-speed shortening, causes changes in thin filament activation at fixed Ca(2+) concentrations that can be followed by recording fluorescence intensity. Upon changing to high-speed shortening we observed small (<20%) changes in fluorescence that became faster at higher Ca(2+) concentrations. At all Ca(2+) concentrations, these changes are more than 10-fold faster than force redevelopment subsequent to the period of unloaded shortening. We interpret this as an indication that equilibration among different states of the thin filament is rapid and becomes faster as Ca(2+) is raised. Fast equilibration suggests that the rate constant of force redevelopment is not limited by changes in the activation level of thin filaments induced by the isotonic contraction before force redevelopment. Instead, our modeling shows that, in agreement with our previous proposal for the regulation of muscle contraction, a rapid and Ca(2+)-dependent equilibration among different states of the thin filament can fully account for the Ca(2+) dependence of force redevelopment and the fluorescence changes described in this study.

Stiffness of skinned rabbit psoas fibers in MgATP and MgPPi solution

The stiffness of single skinned rabbit psoas fibers was measured during rapid length changes applied to one end of the fibers. Apparent fiber stiffness was taken as the initial slope when force was plotted vs. change in sarcomere length. In the presence of MgATP, apparent fiber stiffness increased with increasing speed of stretch. With the fastest possible stretches, the stiffness of relaxed fibers at an ionic strength of 20 mM reached more than 50% of the stiffness measured in rigor. However, it was not clear whether apparent fiber stiffness had reached a maximum, speed independent value. The same behavior was seen at several ionic strengths, with increasing ionic strength leading to a decrease in the apparent fiber stiffness measured at any speed of stretch. A speed dependence of apparent fiber stiffness was demonstrated even more clearly when stiffness was measured in the presence of 4 mM MgPPi. In this case, stiffness varied with speed of stretch over about four decades. This speed dependence of apparent fiber stiffness is likely due to cross-bridges detaching and reattaching during the stiffness measurement (Schoenberg, 1985. Biophys. J. 48:467). This means that obtaining an estimate of the maximum number of cross-bridges attached to actin in relaxed fibers at various ionic strengths is not straightforward. However, the data we have obtained are consistent with other estimates of cross-bridge affinity for actin in fibers (Brenner et al., 1986. Biophys. J. In press.) which suggest that ~60-90% of the cross-bridges attached in rigor are attached in relaxed fibers at an ionic strength of 20 mM and ~2-10% of this number of cross-bridges are attached in a relaxed fiber at an ionic strength of 170 mM.

Thin Filament Activation Probed by Fluorescence of N-((2-(Iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole-Labeled Troponin I Incorporated into Skinned Fibers of Rabbit Psoas Muscle

A method is described for the exchange of native troponin of single rabbit psoas muscle fibers for externally applied troponin complexes without detectable impairment of functional properties of the skinned fibers. This approach is used to exchange native troponin for rabbit skeletal troponin with a fluorescent label (N-((2-(iodoacetoxy)ethyl)-N-methyl)amino-7-nitrobenz-2-oxa-1, 3-diazole, IANBD) on Cys(133) of the troponin I subunit. IANBD-labeled troponin I has previously been used in solution studies as an indicator for the state of activation of reconstituted actin filaments (. Proc. Natl. Acad. Sci. USA. 77:7209-7213). In the skinned fibers, the fluorescence of this probe is unaffected when cross-bridges in their weak binding states attach to actin filaments but decreases either upon the addition of Ca(2+) or when cross-bridges in their strong binding states attach to actin. Maximum reduction is observed when Ca(2+) is raised to saturating concentrations. Additional attachment of cross-bridges in strong binding states gives no further reduction of fluorescence. Attachment of cross-bridges in strong binding states alone (low Ca(2+) concentration) gives only about half of the maximum reduction seen with the addition of calcium. This illustrates that fluorescence of IANBD-labeled troponin I can be used to evaluate thin filament activation, as previously introduced for solution studies. In addition, at nonsaturating Ca(2+) concentrations IANBD fluorescence can be used for straightforward classification of states of the myosin head as weak binding (nonactivating) and strong binding (activating), irrespective of ionic strength or other experimental conditions. Furthermore, the approach presented here not only can be used as a means of exchanging native skeletal troponin and its subunits for a variety of fluorescently labeled or mutant troponin subunits, but also allows the exchange of native skeletal troponin for cardiac troponin.

Theoretical models for cooperative steady-state ATPase activity of myosin subfragment-1 on regulated actin

Recent theoretical work on the cooperative equilibrium binding of myosin subfragment-1-ADP to regulated actin, as influenced by Ca2+, is extended here to the cooperative steady-state ATPase activity of myosin subfragment-1 on regulated actin. Exact solution of the general steady-state problem will require Monte Carlo calculations. Three interrelated special cases are discussed in some detail and sample computer (not Monte Carlo) solutions are given. The eventual objective is to apply these considerations to in vitro experimental data and to in vivo muscle models.

Molecular dynamics studies on troponin (TnI-TnT-TnC) complexes: insight into the regulation of muscle contraction.

Abstract Mutations of any subunit of the troponin complex may lead to serious disorders. Rational approaches to managing these disorders require knowledge of the complex interactions among the three subunits that are required for proper function. Molecular dynamics (MD) simulations were performed for both skeletal (sTn) and cardiac (cTn) troponin. The interactions and correlated motions among the three components of the troponin complex were analyzed using both Molecular Mechanics-Generalized Born Surface Area (MMGBSA) and cross-correlation techniques. The TnTH2 helix was strongly positively correlated with the two long helices of TnI. The C domain of TnC was positively correlated with TnI and TnT. The N domain of TnC was negatively correlated with TnI and TnT in cTn, but not in sTn. The two C-domain calcium-binding sites of TnC were dynamically correlated. The two regulatory N-domain calcium-binding sites of TnC were dynamically correlated, even though the calcium-binding site I is dysfunctional. The strong interaction residue pairs and the strong dynamically correlated residues pairs among the three components of troponin complexes were identified. These correlated motions are consistent with the idea that there is a high degree of cooperativity among the components of the regulatory complex in response to Ca2+ and other effectors. This approach may give insight into the mechanism by which mutations of troponin cause disease. It is interesting that some observed disease causing mutations fall within regions of troponin that are strongly correlated or interacted.

Calponin interaction with alpha-actinin-actin: evidence for a structural role for calponin.

The purpose of this study was to address the paradox of calponin localization with alpha-actinin and filamin, two proteins with tandem calponin homology (CH) domains, by determining the effect of these proteins on the binding of calponin to actin. The results show that actin can accommodate near-saturating concentrations of either calponin and alpha-actinin or calponin and filamin with little change or no change in ligand affinity. Little direct interaction occurred between alpha-actinin and calponin in the absence of actin, so this effect is not likely to explain the co-distribution of these proteins. Calponin, like alpha-actinin, induced elastic gel formation when added to actin. When alpha-actinin was added to newly formed calponin/actin gels, no change was seen in the mechanical properties of the gel compared to calponin and actin alone. However, when calponin was added to newly formed alpha-actinin/actin gels, the resulting gel was much stronger than the gels formed by either ligand alone. Furthermore, gels formed by the addition of calponin to alpha-actinin/actin exhibited a phenomenon known as strain hardening, a characteristic of mechanically resilient gels. These results add weight to the concept that one of the functions of calponin is to stabilize the actin cytoskeleton.

Caldesmon and thin-filament regulation of muscle contraction

Smooth muscle contraction is regulated by phosphorylation of myosin and also possibly by the actin associated protein, caldesmon. The properties of caldesmon are discussed and compared with those of tropomyosin-troponin, the well characterized actin-based regulatory system of striated muscle. Caldesmon functions quite differently from tropomyosin-troponin. Under relaxing conditions tropomyosin-troponin does not affect the binding of myosin subfragment-1 to actin. In contrast, caldesmon strongly inhibits the binding of subfragment-1 to actin in the presence of ATP. This inhibition of binding parallels the decrease in ATPase activity that occurs as the caldesmon concentration is increased. Caldesmon has the opposite effect on the two headed myosin subfragment, heavy meromyosin. The apparent binding of skeletal heavy meromyosin increases slightly as the caldesmon concentration is increased, although the rate of ATP hydrolysis is inhibited. It is suggested that in the presence of caldesmon, myosin·ATP does not bind to the productive actin binding site but interacts with a distinct site on actin-caldesmon. This could lead to both an inhibition of ATP hydrolysis and an inrease in resting stiffness of relaxed smooth muscle.