Dephosphorylation Of Myosin Research Articles

Caldesmon enhances motility inhibition by myosin dephosphorylation.

Some studies have suggested that smooth muscle caldesmon (hCaD) participates in force maintenance at low myosin light chain (LC20) phosphorylation. To elucidate the effects of hCaD on the molecular mechanics of dephosphorylating smooth muscle myosin, we injected myosin light chain phosphatase (MLCP) during in vitro motility measurements performed in the presence of hCaD (400 nM) or its absence. Because hCaD increases the binding force of unphosphorylated smooth muscle myosin to actin, we hypothesized that, upon LC20 dephosphorylation, the fraction (fmot) and the average velocity (vavg) of moving actin filaments would decrease at a greater rate in the presence of hCaD. We fitted sigmoidal curves to fmot and vavg and estimated the rate constant (k) and the midpoint (t0) of the decrease. k was larger in the presence of hCaD (3.6×10−1±6.4×10−2 s−1) than without (1.8×10−1±1.4×10−2 s−1), p = 0.039, n = 5. Additionally, t0 was smaller in the presence of hCaD (32±3 s) than without (44±2 s), p = 0.008, n = 5. These results can be explained either by an increased dephosphorylation rate in the presence of hCaD or by an increase of the reattachment of dephosphorylated myosin to actin. In line with previous experiments that demonstrated that hCaD inhibits the actomyosin ATPase, our sigmoidal fits also show a lower vavg in the presence of hCaD (0.45±0.04 µm/s) than without (0.63±0.06 µm/s), p=0.04, before the addition of MLCP. However, hCaD had no effect on k or t0 of vavg during MLCP injection. The lack of concordance between the fmot and vavg data may be due to the fact that the vavg data are noisier after dephosphorylation. Our results agree with previous data obtained at the whole muscle level that showed that hCaD increases the rate of relaxation of arterial smooth muscle and accelerates the decay in tension of skinned smooth muscle strips.

Striated muscle MLCP is regulated by MYPT2 to limit baseline cardiac myosin phosphorylation.

Cardiac myosin is a hexameric protein comprised of two protamers that each have a heavy chain, a regulatory light chain (RLC), and an essential light chain. Constitutive phosphorylation of cardiac myosin in beating hearts at 0.4 mol phosphate/mol RLC is necessary for normal function. This level is maintained by balanced activities of the cardiac myosin light chain kinase (cMLCK) and myosin light chain phosphatase (MLCP). The canonical MLCP is well-studied in smooth muscles, and is defined as a trimeric holoenzyme comprised of a myosin target regulatory subunit MYPT1, a type-1 catalytic subunit PP1cβ, and an accessory protein M21. In striated muscles, the dominantly expressed regulatory subunit is MYPT2. There is limited information on the localization and regulatory contributions of MYPT2. Studies of regulation of cardiac myosin dephosphorylation is hindered by the fact that myosin dephosphorylation occurs rapidly in isolated cardiac myocytes, and cultured “cardiac” cell lines do not express the regulatory proteins at levels comparable to those of freshly isolated cardiac myocytes. We generated a floxed MYPT2 mouse line and conditionally knocked out nearly all detectable MYPT2 protein in cardiac myocytes, and used this model to determine that: 1) MYPT2 localizes to myosin in vivo, 2) MYPT1 expression does not compensate for the reduction in MYPT2 in the cardiac muscle, 3) the cardiac MLCP holoenzyme expression level is partly regulated by stabilization of the catalytic subunit by MYPT2, 4) without MYPT2-regulated MLCP activity, baseline cardiac RLC phosphorylation is increased to 0.6 mol phosphate/mol RLC, and 5) MYPT2 knockout animals have reduced responses to pressure-overload induced cardiac hypertrophy. These findings help to fill knowledge-gaps about the fundamental properties of the cardiac muscle MLCP.

Myosin light chain phosphatase catalytic subunit dephosphorylates cardiac myosin via mechanisms dependent and independent of the MYPT regulatory subunits

Cardiac muscle myosin regulatory light chain (RLC) is constitutively phosphorylated at ∼0.4 mol phosphate/mol RLC in normal hearts, and phosphorylation is maintained by balanced activities of dedicated cardiac muscle–specific myosin light chain kinase and myosin light chain phosphatase (MLCP). Previously, the identity of the cardiac-MLCP was biochemically shown to be similar to the smooth muscle MLCP, which is a well-characterized trimeric protein comprising the regulatory subunit (MYPT1), catalytic subunit PP1cβ, and accessory subunit M20. In smooth muscles in vivo, MYPT1 and PP1cβ co-stabilize each other and are both necessary for normal smooth muscle contractions. In the cardiac muscle, MYPT1 and MYPT2 are both expressed, but contributions to physiological regulation of cardiac myosin dephosphorylation are unclear. We hypothesized that the main catalytic subunit for cardiac-MLCP is PP1cβ, and maintenance of RLC phosphorylation in vivo is dependent on regulation by striated muscle–specific MYPT2. Here, we used PP1cβ conditional knockout mice to biochemically define cardiac-MLCP proteins and developed a cardiac myofibrillar phosphatase assay to measure the direct contribution of MYPT-regulated and MYPT-independent phosphatase activities toward phosphorylated cardiac myosin. We report that (1) PP1cβ is the main isoform expressed in the cardiac myocyte, (2) cardiac muscle pathogenesis in PP1cβ knockout animals involve upregulation of total PP1cα in myocytes and non-muscle cells, (3) the stability of cardiac MYPT1 and MYPT2 proteins in vivo is not dependent on the PP1cβ expression, and (4) phosphorylated myofibrillar cardiac myosin is dephosphorylated by both myosin-targeted and soluble MYPT-independent PP1cβ activities. These results contribute to our understanding of the cardiac-MLCP in vivo.

PP1cβ dephosphorylates cardiac myosin by MYPT‐dependent and independent mechanisms

In beating hearts, cardiac muscle myosin is phosphorylated at its regulatory light chain (RLC) at 0.4 mol phosphate/mol RLC. This level of constitutive phosphorylation is necessary for normal cardiac function, and is maintained by balanced activities of the dedicated cardiac muscle‐specific myosin light chain kinase (MLCK) and myosin light chain phosphatase (MLCP). The MLCP is best characterized in the smooth muscle, and is comprised of the regulatory myosin phosphatase target subunit MYPT1, a catalytic subunit PP1cβ, and an accessory subunit of unknown function M20. In smooth muscles in vivo, MYPT1 and PP1cβ co‐stabilize each other and are both necessary for normal smooth muscle contractions. In the cardiac muscle, the ubiquitously‐expressed MYPT1 and the striated muscle‐specific MYPT2 are both expressed in the cardiomyocyte, but the contributions to physiological regulation of cardiac myosin dephosphorylation by PP1cβ are unclear.Using a conditional knockout of PP1cβ, we tested the hypothesis that the main catalytic subunit for cardiac MLCP is PP1cβ, and that its activity toward phosphorylated cardiac RLC in vivo is dependent on regulation by striated muscle‐specific MYPT2. We measured the effects of the knockout on the expression levels of other regulators of cardiac RLC phosphorylation, and a cardiac myofibrillar phosphatase assay was developed to measure MYPT‐regulated and MYPT‐independent phosphatase activities toward phosphorylated cardiac myosin. We report that 1) PP1cβ is the main isoform expressed in the cardiac myocyte, 2) cardiac muscle pathogenesis in PP1cβ knockout animals involve upregulation of PP1cα and MYPT1 in activated myofibroblasts, 3) the protein stability of endogenous mouse cardiac MYPT1 and MYPT2 are not dependent on the expression of PP1cβ, and 4) phosphorylated cardiac myosin in intact myofibrillar preparations are dephosphorylated by both myosin‐targeted and soluble MYPT‐independent PP1cβ activities. These results contribute to defining properties of the cardiac MLCP in vivo.

Activation of Myosin Phosphatase by Epigallocatechin-Gallate Sensitizes THP-1 Leukemic Cells to Daunorubicin.

The Myosin Phosphatase (MP) holoenzyme is composed of a Protein Phosphatase type 1 (PP1) catalytic subunit and a regulatory subunit termed Myosin Phosphatase Target subunit 1 (MYPT1). Besides dephosphorylation of myosin, MP has been implicated in the control of cell proliferation via dephosphorylation and activation of the tumor suppressor gene products, retinoblastoma protein (pRb) and merlin. Inhibition of MP was shown to attenuate the drug-induced cell death of leukemic cells by chemotherapeutic agents, while activation of MP might have a sensitizing effect. Recently, Epigallocatechin-Gallate (EGCG), a major component of green tea, was shown to activate MP by inducing the dephosphorylation of MYPT1 at phospho-Thr696 (MYPT1pT696), which might confer enhanced chemosensitivity to cancer cells. THP-1 leukemic cells were treated with EGCG and Daunorubicin (DNR) and cell viability was analyzed. Phosphorylation of tumor suppressor proteins was detected by Western blotting. EGCG or DNR (at sub-lethal doses) alone had moderate effects on cell viability, while the combined treatment caused a significant decrease in the number of viable cells by enhancing apoptosis and decreasing proliferation. EGCG plus DNR decreased the phosphorylation level of MYPT1pT696, which was accompanied by prominent dephosphorylation of pRb. In addition, significant dephosphorylation of merlin was observed when EGCG and DNR were applied together. Our results suggest that EGCG-induced activation of MP might have a regulatory function in mediating the chemosensitivity of leukemic cells via dephosphorylation of tumor suppressor proteins.

Dephosphorylation of Tonic and Phasic Smooth Muscle Myosin in the In Vitro Motility Assay Exhibits Different Kinetics

Tonic smooth muscle has a greater propensity to maintain force during dephosphorylation of its regulatory light chain compared to phasic smooth muscle. To elucidate the underlying mechanism, we injected myosin light chain phosphatase (MLCP) during regular in vitro motility assays (100% tonic or phasic smooth muscle myosin) and mixture assays (25% skeletal: 75% tonic or phasic). Tonic muscle myosin was purified from pig stomach fundus, phasic muscle myosin and MLCP were purified from avian gizzards, and skeletal muscle myosin was purified from chicken breast. In vitro motility assay chambers were devised with a top opening covered with a microporous membrane to allow diffusion of MLCP from injections during the assays. In the regular assay, we observed that the velocity of actin filaments started decreasing before the fraction of moving filaments (fmot) decreased. The time from the start of velocity decrease until near complete stop of the assay (<20% fmot) was 45% greater in tonic compared to phasic muscle myosin. In the mixture assay, we observed that the increase in velocity as a result of a reduction in the number of the slower, active smooth muscle myosin heads took 79% longer for the tonic-skeletal mixture compared to the phasic-skeletal mixture. These data could be explained by a slower rate of dephosphorylation of tonic muscle myosin, caused by either specificity of the MLCP purified from phasic muscle or inherent differences in dephosphorylation rates between tonic and phasic muscle myosin. Alternatively, reduced velocity could be caused by the formation of some slower detaching cross-bridges. These slowly detaching cross-bridges could result from dephosphorylation of myosin while attached to actin, as previously suggested in the latch-state hypothesis. More studies are underway to discriminate between these possibilities.

Abstract 916: Evidence of Cardiac Myosin Dephosphorylation by Two Distinct Pools of Myosin Light Chain Phosphatases

Muscle myosins are hexameric proteins comprised of two each of the heavy chain, essential light chain, and regulatory light chain (RLC). In normal beating hearts, cardiac myosin RLC is phosphorylated, and the extents maintained at ~0.45 mol phosphate/mol RLC by the balanced activities of cardiac-specific myosin light chain kinases (cMLCK) and phosphatases (cMLCP). Hearts in failure have decreased cMLCK expression and RLC phosphorylation, indicating maintenance of near half-maximal RLC phosphorylation levels is necessary for normal function. However, how cMLCK and cMLCP activities are regulated is unclear. MLCP has been biochemically defined as a trimeric protein comprised of a regulatory myosin target subunit (MYPT1 in smooth muscles, and MYPT2 in striated muscles), a catalytic subunit PP1cβ, and a small subunit M20. Interrogations into the specificity and regulatory activity of smooth muscle MLCP using conditional MYPT1 knockout animals have provided evidence of MYPT1-independent RLC phosphatase activity, which contradicts long-standing dogma. Extending upon recent studies of PP1cβ knockout in smooth muscles, we knocked out PP1cβ from the cardiac muscle to test the hypothesis that a distinct MYPT-independent pool of PP1cβ activity directly contributes to cRLC dephosphorylation. Two weeks post gene-ablation, PP1cβ protein was reduced by &gt;60%, and cRLC phosphorylation in beating hearts was increased to 0.6 mol phosphate/mol cRLC, without significant changes in MYPT1 or MYPT2 expression. Additionally, we measured MYPT-dependent and MYPT-independent PP1cβ phosphatase activities toward cRLC dephosphorylation in mouse cardiac myofibrils. These studies provide further evidence that cardiac RLC is dephosphorylated by two distinct pools of MLCP activities.

The role of caldesmon and its phosphorylation by ERK on the binding force of unphosphorylated myosin to actin

BackgroundStudies conducted at the whole muscle level have shown that smooth muscle can maintain tension with low Adenosine triphosphate (ATP) consumption. Whereas it is generally accepted that this property (latch-state) is a consequence of the dephosphorylation of myosin during its attachment to actin, free dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the role of caldesmon (CaD) in regulating the binding force of unphosphorylated tonic smooth muscle myosin to actin. MethodsTo measure the effect of CaD on the binding of unphosphorylated myosin to actin (in the presence of ATP), we used a single beam laser trap assay to quantify the average unbinding force (Funb) in the absence or presence of caldesmon, extracellular signal-regulated kinase (ERK)-phosphorylated CaD, or CaD plus tropomyosin. ResultsFunb from unregulated actin (0.10±0.01pN) was significantly increased in the presence of CaD (0.17±0.02pN), tropomyosin (0.17±0.02pN) or both regulatory proteins (0.18±0.02pN). ERK phosphorylation of CaD significantly reduced the Funb (0.06±0.01pN). Inspection of the traces of the Funb as a function of time suggests that ERK phosphorylation of CaD decreases the binding force of myosin to actin or accelerates its detachment. ConclusionsCaD enhances the binding force of unphosphorylated myosin to actin potentially contributing to the latch-state. ERK phosphorylation of CaD decreases this binding force to very low levels. General significanceThis study suggests a mechanism that likely contributes to the latch-state and that explains the muscle relaxation from the latch-state.

Myosin Phosphatase Isoforms as Determinants of Smooth Muscle Contractile Function and Calcium Sensitivity of Force Production

The dephosphorylation of myosin by the MP causes smooth muscle relaxation. MP is also a key target of signals that regulate vascular tone and thus blood flow and pressure. Here, we review studies from the past two decades that support the hypothesis that the regulated expression of MP subunits is a critical determinant of smooth muscle responses to constrictor and dilator signals. In particular, the highly regulated splicing of the regulatory subunit Mypt1 Exon 24 is proposed to tune sensitivity to NO/cGMP-mediated relaxation. The regulated transcription of the MP inhibitory subunit CPI-17 is proposed to determine sensitivity to agonist-mediated constriction. The expression of these subunits is specific in the microcirculation and varies in developmental and disease contexts. To date, the relationship between MP subunit expression and vascular function in these different contexts is correlative; confirmation of the hypothesis will require the generation of genetically engineered mice to test the role of MP subunits and their isoforms in the specificity of vascular smooth muscle responses to constrictor and dilator signals.

The Role of Caldesmon and Its Phosphorylation by ERK on the Binding Force of Unphosphorylated Myosin to Actin

Smooth muscle has the unique property of maintaining tension with low ATP consumption. It is generally accepted that this property, called the latch-state, results from the dephosphorylation of myosin while attached to actin. However, detached dephosphorylated myosin can also bind to actin and contribute to force maintenance. We investigated the effect of caldesmon on the binding force of unphosphorylated myosin to actin. We quantified the average unbinding force (Funb) in the absence or presence of caldesmon, ERK phosphorylated caldesmon, or caldesmon plus tropomyosin.

Unphosphorylated calponin enhances the binding force of unphosphorylated myosin to actin

BackgroundSmooth muscle has the distinctive ability to maintain force for long periods of time and at low energy costs. While it is generally agreed that this property, called the latch-state, is due to the dephosphorylation of myosin while attached to actin, dephosphorylated-detached myosin can also attach to actin and may contribute to force maintenance. Thus, we investigated the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. MethodsTo measure the effect of calponin on the binding of unphosphorylated myosin to actin, we used the laser trap assay to quantify the average force of unbinding (Funb) in the absence and presence of calponin or phosphorylated calponin. ResultsFunb from F-actin alone (0.12±0.01pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02pN). This enhancement was lost when calponin was phosphorylated (0.12±0.01pN). To further verify that this enhancement of Funb was due to the cross-linking of actin to myosin by calponin, we repeated the measurements at high ionic strength. Indeed, the Funb obtained at a [KCl] of 25mM (0.21±0.02pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01pN). ConclusionsThis study provides direct molecular level-evidence that calponin enhances the binding force of unphosphorylated myosin to actin by cross-linking them and that this is reversed upon calponin phosphorylation. Thus, calponin might play an important role in the latch-state. General significanceThis study suggests a new mechanism that likely contributes to the latch-state, a fundamental and important property of smooth muscle that remains unresolved.

Elevated Expression of the Integrin-Associated Protein PINCH Suppresses the Defects of Drosophila melanogaster Muscle Hypercontraction Mutants

A variety of human diseases arise from mutations that alter muscle contraction. Evolutionary conservation allows genetic studies in Drosophila melanogaster to be used to better understand these myopathies and suggest novel therapeutic strategies. Integrin-mediated adhesion is required to support muscle structure and function, and expression of Integrin adhesive complex (IAC) proteins is modulated to adapt to varying levels of mechanical stress within muscle. Mutations in flapwing (flw), a catalytic subunit of myosin phosphatase, result in non-muscle myosin hyperphosphorylation, as well as muscle hypercontraction, defects in size, motility, muscle attachment, and subsequent larval and pupal lethality. We find that moderately elevated expression of the IAC protein PINCH significantly rescues flw phenotypes. Rescue requires PINCH be bound to its partners, Integrin-linked kinase and Ras suppressor 1. Rescue is not achieved through dephosphorylation of non-muscle myosin, suggesting a mechanism in which elevated PINCH expression strengthens integrin adhesion. In support of this, elevated expression of PINCH rescues an independent muscle hypercontraction mutant in muscle myosin heavy chain, MhcSamba1. By testing a panel of IAC proteins, we show specificity for PINCH expression in the rescue of hypercontraction mutants. These data are consistent with a model in which PINCH is present in limiting quantities within IACs, with increasing PINCH expression reinforcing existing adhesions or allowing for the de novo assembly of new adhesion complexes. Moreover, in myopathies that exhibit hypercontraction, strategic PINCH expression may have therapeutic potential in preserving muscle structure and function.

Drosophila PATJ supports adherens junction stability by modulating Myosin light chain activity

The assembly and consolidation of the adherens junctions (AJs) are key events in the establishment of an intact epithelium. However, AJs are further modified to obtain flexibility for cell migration and morphogenetic movements. Intact AJs in turn are a prerequisite for the establishment and maintenance of apical-basal polarity in epithelial cells. In this study, we report that the conserved PDZ (PSD95, Discs large, ZO-1) domain-containing protein PATJ (Pals1-associated tight junction protein) was not per se crucial for the maintenance of apical-basal polarity in Drosophila melanogaster epithelial cells but rather regulated Myosin localization and phosphorylation. PATJ directly bound to the Myosin-binding subunit of Myosin phosphatase and decreased Myosin dephosphorylation, resulting in activated Myosin. Thereby, PATJ supports the stability of the Zonula Adherens. Notably, weakening of AJ in a PATJ mutant epithelium led first to a loss of Myosin from the AJ, subsequently to a disassembly of the AJ, and finally, to a loss of apical-basal polarity and disruption of the tissue.

Myosin phosphatase is inactivated by caspase-3 cleavage and phosphorylation of myosin phosphatase targeting subunit 1 during apoptosis

In nonapoptotic cells, the phosphorylation level of myosin II is constantly maintained by myosin kinases and myosin phosphatase. During apoptosis, caspase-3-activated Rho-associated protein kinase I triggers hyperphosphorylation of myosin II, leading to membrane blebbing. Although inhibition of myosin phosphatase could also contribute to myosin II phosphorylation, little is known about the regulation of myosin phosphatase in apoptosis. In this study, we have demonstrated that, in apoptotic cells, the myosin-binding domain of myosin phosphatase targeting subunit 1 (MYPT1) is cleaved by caspase-3 at Asp-884, and the cleaved MYPT1 is strongly phosphorylated at Thr-696 and Thr-853, phosphorylation of which is known to inhibit myosin II binding. Expression of the caspase-3 cleaved form of MYPT1 that lacked the C-terminal end in HeLa cells caused the dissociation of MYPT1 from actin stress fibers. The dephosphorylation activity of myosin phosphatase immunoprecipitated from the apoptotic cells was lower than that from the nonapoptotic control cells. These results suggest that down-regulation of MYPT1 may play a role in promoting hyperphosphorylation of myosin II by inhibiting the dephosphorylation of myosin II during apoptosis.

Calponin Cross-Links Unphosphorylated Myosin to Actin and Enhances its Binding Force

Smooth muscle is unique in its capability to maintain force for long periods of time at low ATP (energy) consumption. This property, called the latch-state, is hypothesized to occur due to the dephosphorylation of myosin while attached to actin. Alternative theories have proposed that dephosphorylated-detached myosin can also re-attach to actin and contribute to force maintenance. The goal of this study was to investigate the role of calponin in regulating and enhancing the binding force of unphosphorylated tonic muscle myosin to actin. To confirm the calponin regulation of our actin filaments at physiological concentrations, we used the in-vitro motility assay to measure the rate (1/2max) of actin propulsion by myosin. Our results showed a significant decrease in 1/2max (from 0.54±0.01 μm/s (mean±SE) in the absence to 0.43±0.01 μm/s in the presence of calponin). When calponin was phosphorylated, this inhibitory effect was suppressed (0.50±0.01 μm/s). To measure the force of binding (Funb) of unphosphorylated myosin to actin, we used the laser trap assay. Funb was normalized by the number of myosin molecules estimated per actin filament length. Funb of unregulated actin (0.12±0.01 pN; mean±SE) was significantly increased in the presence of calponin (0.20±0.02 pN). When calponin was phosphorylated, this enhancement was lost (0.12±0.01 pN). To verify whether this enhancement of Funb is due to cross-linking of myosinto actin by calponin, we repeated the measurements at high [KCl], as the calponin affinity for myosin decreases at high ionic strength. Indeed, the Funb, in presence of calponin, obtained at a [KCl] of 25mM (0.21±0.02 pN; mean±SE) was significantly decreased at a [KCl] of 150mM, (0.13±0.01 pN). These data demonstrate that calponin enhances the force of binding of unphosphorylated myosin to actin by cross-linking them together.

A Novel Role of Caldesmon during Smooth Muscle Relaxation

The effect of h-caldesmon (h-CaD) on smooth muscle contractility was examined by force measurements on tissues isolated from wild-type (WT) and h-CaD-null (KO) mice. Both aorta and tail arteries from KO mice relaxed more slowly than that from WT animals. The force decay of beta-escin-permeabilized, kinase-inhibited tissues under pCa>9 was best fitted with a two-exponential process. We found that the apparently slower relaxation for the KO samples was not due to lower rate constants, but rather, was because of increased amplitude of the slower component of the decay. To search for an explanation for the observed relaxation kinetics, we drew some lessons from the work done on striated muscles. Muscle relaxation involves two major events: crossbridge detachment and change of the thin filament from active to inactive state. The latter occurs first, corresponding to the initial lag phase, followed by the rapid force decay, the crossbridge detachment. In smooth muscle, the lag phase is ascribed to myosin dephosphorylation, but it may also include the filament state change, i.e., CaD-tropomyosin moving to a position on actin filaments that hinders myosin binding. This “off-position” is under strain, because the dephosphorylated crossbridges are still attached. Assuming that there are two possible positions for CaD-tropomyosin to occupy, one displacing the bound myosin more promptly than the other, the crossbridge would detach at two different rates, resulting in the observed biphasic decay. Our data then suggest that when h-CaD is absent, fewer tropomyosin molecules are at the blocking position. Thus, the return of smooth muscle tropomyosin to the proper inhibitory position upon relaxation needs help from h-CaD. In striated muscle the movement of tropomyosin was shown to be facilitated by troponin subunits. CaD may play a troponin-like role in smooth muscle both structurally and functionally.

Ablation of smooth muscle caldesmon affects the relaxation kinetics of arterial muscle

Smooth muscle caldesmon (h-CaD) is an actin- and myosin-binding protein that reversibly inhibits the actomyosin ATPase activity in vitro. To test the function of h-CaD in vivo, we eliminated its expression in mice. The h-CaD-null animals appeared normal and fertile, although the litter size was smaller. Tissues from the homozygotes lacked h-CaD and exhibited upregulation of the non-muscle isoform, l-CaD, in visceral, but not vascular tonic smooth muscles. While the Ca(2+) sensitivity of force generation of h-CaD-deficient smooth muscle remained largely unchanged, the kinetic behavior during relaxation in arteries was different. Both intact and permeabilized arterial smooth muscle tissues from the knockout animals relaxed more slowly than those of the wild type. Since this difference occurred after myosin dephosphorylation was complete, the kinetic effect most likely resulted from slower detachment of unphosphorylated crossbridges. Detailed analyses revealed that the apparently slower relaxation of h-CaD-null smooth muscle was due to an increase in the amplitude of a slower component of the biphasic tension decay. While the identity of this slower process has not been unequivocally determined, we propose it reflects a thin filament state that elicits fewer re-attached crossbridges. Our finding that h-CaD modulates the rate of smooth muscle relaxation clearly supports a role in the control of vascular tone.

Drosophila PATJ supports adherens junction stability by modulating Myosin light chain activity

The assembly and consolidation of the adherens junctions (AJs) are key events in the establishment of an intact epithelium. However, AJs are further modified to obtain flexibility for cell migration and morphogenetic movements. Intact AJs in turn are a prerequisite for the establishment and maintenance of apical-basal polarity in epithelial cells. In this study, we report that the conserved PDZ (PSD95, Discs large, ZO-1) domain-containing protein PATJ (Pals1-associated tight junction protein) was not per se crucial for the maintenance of apical-basal polarity in Drosophila melanogaster epithelial cells but rather regulated Myosin localization and phosphorylation. PATJ directly bound to the Myosin-binding subunit of Myosin phosphatase and decreased Myosin dephosphorylation, resulting in activated Myosin. Thereby, PATJ supports the stability of the Zonula Adherens. Notably, weakening of AJ in a PATJ mutant epithelium led first to a loss of Myosin from the AJ, subsequently to a disassembly of the AJ, and finally, to a loss of apical-basal polarity and disruption of the tissue.

Update on Corpus Cavernosum Smooth Muscle Contractile Pathways in Erectile Function: A Role for Testosterone?

Normal erectile function (EF) involves a coordinated relaxation of the arteries that supply the penis and the corpus cavernosum smooth muscle (CCSM), resulting in expansion of the sinusoids and increased intracavernous pressure. But the CCSM spends the majority of its time in the contracted state which is mediated by norepinephrine released from nerve endings and other vasoconstrictors like endothelins released from the endothelium. These agents cause smooth muscle myosin (SMM) phosphorylation by elevating intracellular calcium. When calcium returns to basal levels, the calcium sensitivity increases and prevents myosin dephosphorylation, which involves the RhoA/Rho-kinase (ROK) mechanism, thus maintaining force. Although mounting evidences demonstrate that androgens have a major influence on EF that is not just centrally mediated, this notion remains quite controversial. To summarize the current knowledge on CCSM contractile pathways, the role they play in modulating EF, and the influence of androgens. The article reviews the literature and contains some previously unpublished data on CCSM contraction signaling including the role that androgens are known to play in modulating these pathways. Data from peer-reviewed publications and previously unpublished observations. In addition to downregulation of many pro-erectile molecular mechanisms, decreased testosterone (T) levels upregulate CCSM contractility, including hyperresponsiveness to α-adrenergic agonists, increased SMM phosphorylation, alteration of SMM isoform composition, activation of RhoA/ROK signaling and modulation of sphingosine-1-phosphate regulation of CCSM tone. Decreased T levels upregulate CCSM contractile signaling. Meanwhile, it downregulates CCSM relaxation pathways synergizing to produce erectile dysfunction (ED). Although some urologists and researchers are still skeptical of the influence of androgens on penile erection, understanding these molecular control mechanisms as well as the influence that androgens have on these pathways should provide new evidence supporting the roles of androgens in EF and enhance the discovery of novel targets for drug development to treat ED.

Dictyostelium huntingtin controls chemotaxis and cytokinesis through the regulation of myosin II phosphorylation

Abnormalities in the huntingtin protein (Htt) are associated with Huntington's disease. Despite its importance, the function of Htt is largely unknown. We show that Htt is required for normal chemotaxis and cytokinesis in Dictyostelium discoideum. Cells lacking Htt showed slower migration toward the chemoattractant cAMP and contained lower levels of cortical myosin II, which is likely due to defects in dephosphorylation of myosin II mediated by protein phosphatase 2A (PP2A). htt(-) cells also failed to maintain myosin II in the cortex of the cleavage furrow, generating unseparated daughter cells connected through a thin cytoplasmic bridge. Furthermore, similar to Dictyostelium htt(-) cells, siRNA-mediated knockdown of human HTT also decreased the PP2A activity in HeLa cells. Our data indicate that Htt regulates the phosphorylation status of myosin II during chemotaxis and cytokinesis through PP2A.