Alexander S. Khromov

Contractile properties and proteins of smooth muscles of a calponin knockout mouse

1 The role of h1‐calponin in regulating the contractile properties of smooth muscle was investigated in bladder and vas deferens of mice carrying a targeted mutation in both alleles designed to inactivate the basic calponin gene. These calponin knockout (KO) mice displayed no detectable h1‐calponin in their smooth muscles. 2 The amplitudes of Ca2+ sensitization, force and Ca2+ sensitivity were not significantly different in permeabilized smooth muscle of KO compared with wild‐type (WT) mice, nor were the delays in onset and half‐times of Ca2+ sensitization, initiated by flash photolysis of caged GTPγS, different. 3 The unloaded shortening velocity (Vus) of thiophosphorylated fibres was significantly (P < 0.05) faster in the smooth muscle of KO than WT animals, but could be slowed by exogenous calponin to approximate WT levels; the concentration dependence of exogenous calponin slowing of Vus was proportional to its actomyosin binding in situ. 4 Actin expression was reduced by 25‐50%, relative to that of myosin heavy chain, in smooth muscle of KO mice, without any change in the relative distribution of the actin isoforms. 5 We conclude that the faster Vus of smooth muscle of the KO mouse is consistent with, but does not prove without further study, physiological regulation of the crossbridge cycle by calponin. Our results show no detectable role of calponin in the signal transduction of the Ca2+‐sensitization pathways in smooth muscle.

Phosphorylation-dependent Autoinhibition of Myosin Light Chain Phosphatase Accounts for Ca2+ Sensitization Force of Smooth Muscle Contraction

The reversible regulation of myosin light chain phosphatase (MLCP) in response to agonist stimulation and cAMP/cGMP signals plays an important role in the regulation of smooth muscle (SM) tone. Here, we investigated the mechanism underlying the inhibition of MLCP induced by the phosphorylation of myosin phosphatase targeting subunit (MYPT1), a regulatory subunit of MLCP, at Thr-696 and Thr-853 using glutathione S-transferase (GST)-MYPT1 fragments having the inhibitory phosphorylation sites. GST-MYPT1 fragments, including only Thr-696 and only Thr-853, inhibited purified MLCP (IC50 = 1.6 and 60 nm, respectively) when they were phosphorylated with RhoA-dependent kinase (ROCK). The activities of isolated catalytic subunits of type 1 and type 2A phosphatases (PP1 and PP2A) were insensitive to either fragment. Phospho-GST-MYPT1 fragments docked directly at the active site of MLCP, and this was blocked by a PP1/PP2A inhibitor microcystin (MC)-LR or by mutation of the active sites in PP1. GST-MYPT1 fragments induced a contraction of β-escin-permeabilized ileum SM at constant pCa 6.3 (EC50 = 2 μm), which was eliminated by Ala substitution of the fragment at Thr-696 or by ROCK inhibitors or 8Br-cGMP. GST-MYPT1-(697–880) was 5-times less potent than fragments including Thr-696. Relaxation induced by 8Br-cGMP was not affected by Ala substitution at Ser-695, a known phosphorylation site for protein kinase A/G. Thus, GST-MYPT1 fragments are phosphorylated by ROCK in permeabilized SM and mimic agonist-induced inhibition and cGMP-induced activation of MLCP. We propose a model in which MYPT1 phosphorylation at Thr-696 and Thr-853 causes an autoinhibition of MLCP that accounts for Ca2+ sensitization of smooth muscle force.

The cAMP-responsive Rap1 Guanine Nucleotide Exchange Factor, Epac, Induces Smooth Muscle Relaxation by Down-regulation of RhoA Activity

Agonist activation of the small GTPase, RhoA, and its effector Rho kinase leads to down-regulation of smooth muscle (SM) myosin light chain phosphatase activity, an increase in myosin light chain (RLC20) phosphorylation and force. Cyclic nucleotides can reverse this process. We report a new mechanism of cAMP-mediated relaxation through Epac, a GTP exchange factor for the small GTPase Rap1 resulting in an increase in Rap1 activity and suppression of RhoA activity. An Epac-selective cAMP analog, 8-pCPT-2′-O-Me-cAMP (“007”), significantly reduced agonist-induced contractile force, RLC20, and myosin light chain phosphatase phosphorylation in both intact and permeabilized vascular, gut, and airway SMs independently of PKA and PKG. The vasodilator PGI2 analog, cicaprost, increased Rap1 activity and decreased RhoA activity in intact SMs. Forskolin, phosphodiesterase inhibitor isobutylmethylxanthine, and isoproterenol also significantly increased Rap1-GTP in rat aortic SM cells. The PKA inhibitor H89 was without effect on the 007-induced increase in Rap1-GTP. Lysophosphatidic acid-induced RhoA activity was reduced by treatment with 007 in WT but not Rap1B null fibroblasts, consistent with Epac signaling through Rap1B to down-regulate RhoA activity. Isoproterenol-induced increase in Rap1 activity was inhibited by silencing Epac1 in rat aortic SM cells. Evidence is presented that cooperative cAMP activation of PKA and Epac contribute to relaxation of SM. Our findings demonstrate a cAMP-mediated signaling mechanism whereby activation of Epac results in a PKA-independent, Rap1-dependent Ca2+ desensitization of force in SM through down-regulation of RhoA activity. Cyclic AMP inhibition of RhoA is mediated through activation of both Epac and PKA.

Myosin Essential Light Chain Isoforms Modulate the Velocity of Shortening Propelled by Nonphosphorylated Cross-bridges

The differential effects of essential light chain isoforms (LC17a and LC17b) on the mechanical properties of smooth muscle were determined by exchanging recombinant for endogenous LC17 in permeabilized smooth muscle treated with trifluoperazine (TFP). Co-precipitation with endogenous myosin heavy chain verified that 40–60% of endogenous LC17a could be exchanged for recombinant LC17aor LC17b. Upon addition of MgATP in Ca2+-free solution, recombinant LC17 exchange induced slow contractions unaccompanied by regulatory light chain (RLC) phosphorylation only in TFP-treated, but not in untreated, permeabilized smooth muscle; the shortening velocity and rate of force development were approximately 1.5 and 2 times faster, respectively, in response to LC17a than LC17b. Additional incubation with recombinant, thiophosphorylated RLC increased the shortening velocity, independent of the LC17 isoform exchanged. The LC17-induced contractions of TFP-treated muscles were abolished by prior addition of nonphosphorylated RLC. We suggest that LC17 stiffens the lever arm of myosin and, in the absence of regulation by RLC, permits cross-bridge cycling without requiring RLC phosphorylation. Our results are compatible with nonphosphorylated RLC acting as a repressor and with LC17isoforms modulating the MgADP affinity and, consequently, rate of cooperative cycling of nonphosphorylated cross-bridges.

NUCLEOTIDE BINDING BY ACTOMYOSIN AS A DETERMINANT OF RELAXATION KINETICS OF RABBIT PHASIC AND TONIC SMOOTH MUSCLE

1. The apparent second‐order rate constants (k+T) of ATP‐induced cross‐bridge detachment from rigor in the absence of Ca2+ were determined with laser flash photolysis of caged ATP (cATP) in alpha‐toxin‐permeabilized tonic, rabbit femoral artery and phasic, rabbit bladder smooth muscles. The potential effect of cATP binding to actomyosin (AM) on cross‐bridge kinetics was examined by varying the initial concentration of cATP 2‐fold. For a given [ATP] released from either 10 or 5 mM cATP, the kinetics of relaxation were not significantly different; the estimated dissociation constant for cATP binding to smooth muscle AM was 1‐3 mM. 2. k+T was significantly higher ((9.5 +/‐ 1.3) x 10(4) M‐1 s‐1) in the phasic than in the tonic ((3.0 +/‐ 1.0) x 10(4) M‐1 s‐1) smooth muscle. 3. We conclude that the combination of the significantly lower (approximately 3 times) apparent second‐order rate constant of MgATP association with the approximately 5 times higher affinity of cross‐bridges for MgADP in tonic, than in phasic, smooth muscle is a major determinant of the slower kinetics of relaxation and, probably, shortening velocity of tonic smooth muscle.

Cep57, a multidomain protein with unique microtubule and centrosomal localization domains.

The present study demonstrates different functional domains of a recently described centrosomal protein, Cep57 (centrosomal protein 57). Endogenous Cep57 protein and ectopic expression of full-length protein or the N-terminal coiled-coil domain localize to the centrosome internal to gamma-tubulin, suggesting that it is either on both centrioles or on a centromatrix component. The N-terminus can also multimerize with the N-terminus of other Cep57 molecules. The C-terminus contains a second coiled-coil domain that directly binds to MTs (microtubules). This domain both nucleates and bundles MTs in vitro. This activity was also seen in vivo, as overexpression of full-length Cep57 or the C-terminus generates nocodazole-resistant MT cables in cells. Based on the present findings, we propose that Cep57 serves as a link with its N-terminus anchored to the centriole or centromatrix and its C-terminus to MTs.

Cryo-atomic Force Microscopy of Unphosphorylated and Thiophosphorylated Single Smooth Muscle Myosin Molecules

The purpose of this study was to determine whether steric blockage of one head by the second head of native two-headed myosin was responsible for the inactivity of nonphosphorylated two-headed myosin compared with the high activity of single-headed myosin, as suggested on the basis of electron microscopy of two-dimensional crystals of heavy meromyosin (Wendt, T., Taylor, D., Messier, T., Trybus, K. M., and Taylor, K. A. (1999) J. Cell Biol. 147, 1385–1390; and Wendt, T., Taylor, D., Trybus, K. M., and Taylor, K. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 4361–4366). Our earlier cryo-atomic force microscopy (cryo-AFM) (Zhang, Y., Shao, Z., Somlyo, A. P., and Somlyo, A. V. (1997) Biophys. J. 72, 1308–1318) indicates that thiophosphorylation of the regulatory light chain increases the separation of the two heads of a single myosin molecule, but the thermodynamic probability of steric hindrance by strong binding between the two heads was not determined. We now report this probability determined by cryo-AFM of single whole myosin molecules shown to have normal low ATPase activity (0.007 s–1). We found that the thermodynamic probability of the relative head positions of nonphosphorylated myosin was approximately equal between separated heads as compared with closely apposed heads (energy difference of 0.24 kT (where k is a Boltzman constant and T is the absolute temperature)), and thiophosphorylation increased the number of molecules having separated heads (energy advantage of –1.2 kT (where k is a Boltzman constant and I is the absolute temperature)). Our results do not support the suggestion that strong binding of one head to the other stabilizes the blocked conformation against thermal fluctuations resulting in steric blockage that can account for the low activity of nonphosphorylated two-headed myosin.

Thiophosphorylation of myosin light chain increases rigor stiffness of rabbit smooth muscle

1 The effect of thiophosphorylation of the regulatory myosin light chain (MLC20) on rigor stiffness was determined in permeabilized rabbit bladder smooth muscle. 2 Rigor stiffness of α‐toxin‐permeabilized smooth muscle was significantly increased by thiophosphorylation of MLC20. This increase may have been due to partial shortening (melting) in the proximal rod region and/or stiffening of the regulatory domain of the myosin head. 3 We suggest that phosphorylation of MLC20, by increasing the stiffness of the S1 lever arm and/or S2 hinge regions of the myosin molecule, favours separation of the two phosphorylated heads and consequent deinhibition of motor domain activity.

Molecular Mechanism of Telokin-mediated Disinhibition of Myosin Light Chain Phosphatase and cAMP/cGMP-induced Relaxation of Gastrointestinal Smooth Muscle

Background: Phospho-telokin is a cyclic nucleotide-dependent protein kinase substrate that leads to smooth muscle relaxation. Results: Phospho-telokin activates inhibited phosphorylated myosin phosphatase. Conclusion: Phospho-telokin binds to the phosphorylated myosin phosphatase facilitating its binding to phosphomyosin and myosin dephosphorylation. Significance: This mechanism may play a role in cyclic nucleotide-mediated relaxation of telokin expressing smooth muscles in the gut and vasculature. Phospho-telokin is a target of elevated cyclic nucleotide concentrations that lead to relaxation of gastrointestinal and some vascular smooth muscles (SM). Here, we demonstrate that in telokin-null SM, both Ca2+-activated contraction and Ca2+ sensitization of force induced by a GST-MYPT1(654–880) fragment inhibiting myosin light chain phosphatase were antagonized by the addition of recombinant S13D telokin, without changing the inhibitory phosphorylation status of endogenous MYPT1 (the regulatory subunit of myosin light chain phosphatase) at Thr-696/Thr-853 or activity of Rho kinase. Cyclic nucleotide-induced relaxation of force in telokin-null ileum muscle was reduced but not correlated with a change in MYPT1 phosphorylation. The 40% inhibited activity of phosphorylated MYPT1 in telokin-null ileum homogenates was restored to nonphosphorylated MYPT1 levels by addition of S13D telokin. Using the GST-MYPT1 fragment as a ligand and SM homogenates from WT and telokin KO mice as a source of endogenous proteins, we found that only in the presence of endogenous telokin, thiophospho-GST-MYPT1 co-precipitated with phospho-20-kDa myosin regulatory light chain 20 and PP1. Surface plasmon resonance studies showed that S13D telokin bound to full-length phospho-MYPT1. Results of a protein ligation assay also supported interaction of endogenous phosphorylated MYPT1 with telokin in SM cells. We conclude that the mechanism of action of phospho-telokin is not through modulation of the MYPT1 phosphorylation status but rather it contributes to cyclic nucleotide-induced relaxation of SM by interacting with and activating the inhibited full-length phospho-MYPT1/PP1 through facilitating its binding to phosphomyosin and thus accelerating 20-kDa myosin regulatory light chain dephosphorylation.

Photolytic Release of MgADP Reduces Rigor Force in Smooth Muscle

Photolytic release of MgADP (25-300 microM) from caged ADP in permeabilized tonic (rabbit femoral artery-Rfa) and phasic (rabbit bladder-Rbl) smooth muscle in high-tension rigor state, in the absence of Ca(2+), caused an exponential decline (approximately 1.5% in Rfa and approximately 6% in Rbl) of rigor force, with the rate proportional to the liberated [MgADP]. The apparent second-order rate constant of MgADP binding was estimated as approximately 1.0 x 10(6) M(-1) s(-1) for both smooth muscles. In control experiments, designed to test the specificity of MgADP, photolysis of caged ADP in the absence of Mg(2+) did not decrease rigor force in either smooth muscle, but rigor force decreased after photolytic release of Mg(2+) in the presence of ADP. The effects of photolysis of caged ADP were similar in smooth muscles containing thiophosphorylated or non-phosphorylated regulatory myosin light chains. Stretching or releasing (within range of 0.1-1.2% of initial Ca(2+)-activated force) did not affect the rate or relative amplitude of the force decrease. The effect of additions of MgADP to rigor cross-bridges could result from rotation of the lever arm of smooth muscle myosin, but this need not imply that ADP-release is a significant force-producing step of the physiological cross-bridge cycle.