The Myosin-binding Protein C Motif Binds to F-actin in a Phosphorylation-sensitive Manner

Cardiac myosin-binding protein C (cMyBP-C) is a regulatory protein expressed in cardiac sarcomeres that is known to interact with myosin, titin, and actin. cMyBP-C modulates actomyosin interactions in a phosphorylation-dependent way, but it is unclear whether interactions with myosin, titin, or actin are required for these effects. Here we show using cosedimentation binding assays, that the 4 N-terminal domains of murine cMyBP-C (i.e. C0-C1-m-C2) bind to F-actin with a dissociation constant (K_d) of ∼10 μm and a molar binding ratio (B_max) near 1.0, indicating 1:1 (mol/mol) binding to actin. Electron microscopy and light scattering analyses show that these domains cross-link F-actin filaments, implying multiple sites of interaction with actin. Phosphorylation of the MyBP-C regulatory motif, or m-domain, reduced binding to actin (reduced B_max) and eliminated actin cross-linking. These results suggest that the N terminus of cMyBP-C interacts with F-actin through multiple distinct binding sites and that binding at one or more sites is reduced by phosphorylation. Reversible interactions with actin could contribute to effects of cMyBP-C to increase cross-bridge cycling.Cardiac myosin-binding protein C (cMyBP-C)² is a thick filament accessory protein that performs both structural and regulatory functions within vertebrate sarcomeres. Both roles are likely to be essential in deciphering how a growing number of mutations found in the cMyBP-C gene, i.e. MYBPC3, lead to cardiomyopathies and heart failure in a substantial number of the world''s population (1, 2).Considerable progress has recently been made in determining the regulatory functions of cMyBP-C and it is now apparent that cMyBP-C normally limits cross-bridge cycling kinetics and is critical for cardiac function (3-5). Phosphorylation of cMyBP-C is essential for its regulatory effects because elimination of phosphorylation sites (serine to alanine substitutions) abolishes the ability of protein kinase A (PKA) to accelerate cross-bridge cycling kinetics and blunts cardiac responses to inotropic stimuli (6). The substitutions further impair cardiac function, reduce contractile reserve, and cause cardiac hypertrophy in transgenic mice (6, 7). By contrast, substitution of aspartic acids at these sites to mimic constitutive phosphorylation is benign or cardioprotective (8).Although a role for cMyBP-C in modulating cross-bridge kinetics is supported by several transgenic and knock-out mouse models (6, 7, 9, 10), the precise mechanisms by which cMyBP-C exerts these effects are not completely understood. For instance, the unique regulatory motif or “m-domain” of cMyBP-C binds to the S2 subfragment of myosin in vitro (11) and binding is abolished by PKA-mediated phosphorylation of the m-domain (12). These observations have led to the idea that (un)binding of the m-domain from myosin S2 mediates PKA-induced increases in cross-bridge cycling kinetics. Consistent with this idea, Calaghan and colleagues (13) showed that S2 added to transiently permeabilized myocytes increased their contractility, presumably because added S2 displaced cMyBP-C from binding endogenous S2. However, other reports indicate that cMyBP-C can influence actomyosin interactions through mechanisms unrelated to S2 binding, because either purified cMyBP-C (14) or recombinant N-terminal domains of cMyBP-C (15) affected acto-S1 filament sliding velocities and ATPase rates in the absence of myosin S2. These results thus raise the possibility that interactions with ligands other than myosin S2, such as actin or myosin S1, contribute to effects of cMyBP-C on cross-bridge interaction kinetics.The idea that cMyBP-C interacts with actin to influence cross-bridge cycling kinetics is supported by several studies that implicate the regulatory m-domain or sequences near it in actin binding (16-19). cMyBP-C is a member of the immunoglobulin (Ig) superfamily of proteins and consists of 11 repeating domains that bear homology to either Ig or fibronectin-like folds. Domains are numbered sequentially from the N terminus of cMyBP-C as C0 through C10. The m-domain, a unique sequence of ∼100 amino acids, is located between domains C1 and C2 and is phosphorylated on at least 3 serine residues by PKA (12). Although the precise structure of the m-domain is not known, small angle x-ray scattering data suggest that it is compact and folded in solution and is thus similar in size and dimensions to the surrounding Ig domains (20). Recombinant proteins encompassing the m-domain and/or a combination of adjacent domains including C0, C1, C2, and a proline-alanine-rich sequence that links C0 to C1 have been shown to bind actin (16, 18, 19).The purpose of the present study was to characterize binding interactions of the N terminus of cMyBP-C with actin and to determine whether interactions with actin are influenced by phosphorylation of the m-domain. Results demonstrate that the N terminus of cMyBP-C binds to F-actin and to native thin filaments with affinities similar to that reported for cMyBP-C binding to myosin S2 (11). Furthermore, actin binding was reduced by m-domain phosphorylation, suggesting that reversible interactions of cMyBP-C with actin could contribute to modulation of cross-bridge kinetics.     

Identification of Cardiac Myosin-binding Protein C as a Candidate Biomarker of Myocardial Infarction by Proteomics Analysis

Acute myocardial infarction (AMI) is a common cause of death for which effective treatments are available provided that diagnosis is rapid. The current diagnostic gold standards are circulating cardiac troponins I and T. However, their slow release delays diagnosis, and their persistence limits their utility in the identification of reinfarction. The aim was to identify candidate biomarkers of AMI. Isolated mouse hearts were perfused with oxygenated protein-free buffer, and coronary effluent was collected after ischemia or during matched normoxic perfusion. Effluents were analyzed using proteomics approaches based on one- or two-dimensional initial separation. Of the 459 proteins identified after ischemia with one-dimensional separation, 320 were not detected in the control coronary effluent. Among these were all classic existing biomarkers of AMI. We also identified the cardiac isoform of myosin-binding protein C in its full-length form and as a 40-kDa degradation product. This protein was not detected in the other murine organs examined, increased markedly with even trivial myocardial infarction, and could be detected in the plasma after myocardial infarction in vivo, a profile compatible with a biomarker of AMI. Two-dimensional fluorescence DIGE of ischemic and control coronary effluents identified more than 200 asymmetric spots verified by swapping dyes. Once again existing biomarkers of injury were confirmed as well as posttranslational modifications of antioxidant proteins such as peroxiredoxins. Perfusing hearts with protein-free buffers provides a platform of graded ischemic injury that allows detailed analysis of protein release and identification of candidate cardiac biomarkers like myosin-binding protein C.Acute myocardial infarction (AMI)1 is a common cause of death for which effective treatments are available provided that the condition is rapidly diagnosed. The modern diagnosis of AMI relies on the rise and fall of a specific serum biomarker accompanied by an appropriate circumstance such as chest pain or revascularization. In this accepted paradigm, the diagnosis cannot be ruled in or ruled out without the definite presence or definite absence of a serum biomarker. The ideal biomarker of cardiac injury should be cardiac specific and released rapidly after myocardial injury in direct proportion to the extent of damage. Furthermore, the biomarker should have a high sensitivity and specificity (1). Several biomarkers of AMI have been described in the literature, but only a few, none of which are ideal, have found their way into routine clinical practice. For example, CK-MB starts to increase 4–8 h after coronary artery occlusion and returns to base line within 2–3 days (2). However, its use is limited by its presence in skeletal muscle and normal serum and by sensitivity of the assay to interference, causing some to question its utility (3). Myoglobin is another cytoplasmic protein found in cardiac and skeletal, but not smooth, muscle. It is released even earlier within 1–2 h of AMI and peaks within 5–6 h (2). Unfortunately, any injury to skeletal muscle also causes elevated levels of myoglobin, reducing specificity. Fatty acid-binding proteins (FABPs) are small (15-kDa) cytoplasmic proteins expressed in all tissues with active fatty acid metabolism. Among the nine proteins, heart-specific FABP (H-FABP) is found in heart but also kidney, brain, skeletal muscle, and placenta (4). Following acute myocardial infarction, H-FABP can be detected within 20 min and peaks at 4 h, considerably faster even than CK/CK-MB in the same patient cohort. Although H-FABP concentrations in normal plasma are low, they are known to rise nonspecifically during physical exertion (without a troponin rise), kidney injury, and stroke (5).The most specific and sensitive cardiac proteins released after acute myocardial infarction are cardiac troponins I and T. Both troponins I and T are released slowly, peaking ∼18 h after myocardial infarction, and remain elevated for 7–10 days (2). This slow release is likely the result of their relatively inaccessible cellular location compared with CK-MB, myoglobin, and H-FABP. Troponins regulate the physical interaction of actin and myosin and thus are found almost entirely associated within the crystalline structure of the sarcomere of striated muscle cells (6). The troponin complex is composed of three forms: I, T, and C. Troponins I and T exist as cardiac specific isoforms with epitopes that differ from the corresponding skeletal isoforms. In addition, the absent or extremely low normal circulating levels of troponin provide the greatest dynamic range of any of the currently available biomarkers (7). Although there is no doubt troponins have revolutionized the detection and management of patients with AMI (8), they do have disadvantages. The slow release of troponin delays diagnosis and the initiation of specific treatments that could salvage heart tissue in those in whom it is raised. Similarly, patients in whom it is absent and who are ultimately reassured and discharged are admitted to the hospital unnecessarily. Furthermore, the persistence of troponins limits their utility in the diagnosis of reinfarction.It is therefore widely accepted that there is a need for new biomarkers that can diagnose AMI earlier during its natural history and/or that have a short plasma half-life, allowing use in diagnosis and quantification of reinfarction. The purpose of this study was to use the platform of the crystalloid perfused mouse hearts to perform a systematic proteomics analysis of the coronary effluent after minimal AMI to identify new potential biomarkers (9).     

N-Terminal Domains of Cardiac Myosin Binding Protein C Cooperatively Activate the Thin Filament

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Myocardial Infarction-induced N-terminal Fragment of Cardiac Myosin-binding Protein C (cMyBP-C) Impairs Myofilament Function in Human Myocardium

Myocardial infarction (MI) is associated with depressed cardiac contractile function and progression to heart failure. Cardiac myosin-binding protein C, a cardiac-specific myofilament protein, is proteolyzed post-MI in humans, which results in an N-terminal fragment, C0-C1f. The presence of C0-C1f in cultured cardiomyocytes results in decreased Ca²⁺ transients and cell shortening, abnormalities sufficient for the induction of heart failure in a mouse model. However, the underlying mechanisms remain unclear. Here, we investigate the association between C0-C1f and altered contractility in human cardiac myofilaments in vitro. To accomplish this, we generated recombinant human C0-C1f (hC0C1f) and incorporated it into permeabilized human left ventricular myocardium. Mechanical properties were studied at short (2 μm) and long (2.3 μm) sarcomere length (SL). Our data demonstrate that the presence of hC0C1f in the sarcomere had the greatest effect at short, but not long, SL, decreasing maximal force and myofilament Ca²⁺ sensitivity. Moreover, hC0C1f led to increased cooperative activation, cross-bridge cycling kinetics, and tension cost, with greater effects at short SL. We further established that the effects of hC0C1f occur through direct interaction with actin and α-tropomyosin. Our data demonstrate that the presence of hC0C1f in the sarcomere is sufficient to induce depressed myofilament function and Ca²⁺ sensitivity in otherwise healthy human donor myocardium. Decreased cardiac function post-MI may result, in part, from the ability of hC0C1f to bind actin and α-tropomyosin, suggesting that cleaved C0-C1f could act as a poison polypeptide and disrupt the interaction of native cardiac myosin-binding protein C with the thin filament.     

The Motif of Human Cardiac Myosin-binding Protein C Is Required for Its Ca2+-dependent Interaction with Calmodulin

The N-terminal modules of cardiac myosin-binding protein C (cMyBP-C) play a regulatory role in mediating interactions between myosin and actin during heart muscle contraction. The so-called "motif," located between the second and third immunoglobulin modules of the cardiac isoform, is believed to modulate contractility via an "on-off" phosphorylation-dependent tether to myosin ΔS2. Here we report a novel Ca(2+)-dependent interaction between the motif and calmodulin (CaM) based on the results of a combined fluorescence, NMR, and light and x-ray scattering study. We show that constructs of cMyBP-C containing the motif bind to Ca(2+)/CaM with a moderate affinity (K(D) ～10 μm), which is similar to the affinity previously determined for myosin ΔS2. However, unlike the interaction with myosin ΔS2, the Ca(2+)/CaM interaction is unaffected by substitution with a triphosphorylated motif mimic. Further, Ca(2+)/CaM interacts with the highly conserved residues (Glu(319)-Lys(341)) toward the C-terminal end of the motif. Consistent with the Ca(2+) dependence, the binding of CaM to the motif is mediated via the hydrophobic clefts within the N- and C-lobes that are known to become more exposed upon Ca(2+) binding. Overall, Ca(2+)/CaM engages with the motif in an extended clamp configuration as opposed to the collapsed binding mode often observed in other CaM-protein interactions. Our results suggest that CaM may act as a structural conduit that links cMyBP-C with Ca(2+) signaling pathways to help coordinate phosphorylation events and synchronize the multiple interactions between cMyBP-C, myosin, and actin during the heart muscle contraction.     

Cardiac Myosin-binding protein C modulates the tuning of the molecular motor in the heart

Cardiac myosin binding protein C (cMyBP-C) is an important regulator of cardiac contractility. Its precise effect on myosin cross-bridges (CBs) remains unclear. Using a cMyBP-C^−/− mouse model, we determined how cMyBP-C modulates the cyclic interaction of CBs with actin. From papillary muscle mechanics, CB characteristics were provided using A. F. Huxley's equations. The probability of myosin being weakly bound to actin was higher in cMyBP-C^−/− than in cMyBP-C^+/+. However, the number of CBs in strongly bound, high-force generated state and the force generated per CB were lower in cMyBP-C^−/−. Overall CB cycling and the velocity of CB tilting were accelerated in cMyBP-C^−/−. Taking advantage of the presence of cMyBP-C in cMyBP-C^+/+ myosin solution but not in cMyBP-C^−/−, we also analyzed the effects of cMyBP-C on the myosin-based sliding velocity of actin filaments. At baseline, sliding velocity and the relative isometric CB force, as determined by the amount of α-actinin required to arrest thin filament motility, were lower in cMyBP-C^−/− than in cMyBP-C^+/+. cAMP-dependent protein kinase-mediated cMyBP-C phosphorylation further increased the force produced by CBs. We conclude that cMyBP-C prevents inefficient, weak binding of the myosin CB to actin and has a critical effect on the power-stroke step of the myosin molecular motor.     

Cardiac Fibroblasts Regulate Sympathetic Nerve Sprouting and Neurocardiac Synapse Stability

Sympathetic nervous system (SNS) plays a key role in cardiac homeostasis and its deregulations always associate with bad clinical outcomes. To date, little is known about molecular mechanisms regulating cardiac sympathetic innervation. The aim of the study was to determine the role of fibroblasts in heart sympathetic innervation. RT-qPCR and western-blots analysis performed in cardiomyocytes and fibroblasts isolated from healthy adult rat hearts revealed that Pro-Nerve growth factor (NGF) and pro-differentiating mature NGF were the most abundant neurotrophins expressed in cardiac fibroblasts while barely detectable in cardiomyocytes. When cultured with cardiac fibroblasts or fibroblast-conditioned medium, PC12 cells differentiated into/sympathetic-like neurons expressing axonal marker Tau-1 at neurites in contact with cardiomyocytes. This was prevented by anti-NGF blocking antibodies suggesting a paracrine action of NGF secreted by fibroblasts. When co-cultured with cardiomyocytes to mimic neurocardiac synapse, differentiated PC12 cells exhibited enhanced norepinephrine secretion as quantified by HPLC compared to PC12 cultured alone while co-culture with fibroblasts had no effect. However, when supplemented to PC12-cardiomyocytes co-culture, fibroblasts allowed long-term survival of the neurocardiac synapse. Activated fibroblasts (myofibroblasts) isolated from myocardial infarction rat hearts exhibited significantly higher mature NGF expression than normal fibroblasts and also promoted PC12 cells differentiation. Within the ischemic area lacking cardiomyocytes and neurocardiac synapses, tyrosine hydroxylase immunoreactivity was increased and associated with local anarchical and immature sympathetic hyperinnervation but tissue norepinephrine content was similar to that of normal cardiac tissue, suggesting depressed sympathetic function. Collectively, these findings demonstrate for the first time that fibroblasts are essential for the setting of cardiac sympathetic innervation and neurocardiac synapse stability. They also suggest that neurocardiac synapse functionality relies on a triptych with tight interaction between sympathetic nerve endings, cardiomyocytes and fibroblasts. Deregulations of this triptych may be involved in pathophysiology of cardiac diseases.     

JAK2 and SHP2 Reciprocally Regulate Tyrosine Phosphorylation and Stability of Proapoptotic Protein ASK1

Previously we have shown that tyrosine 718 of ASK1 when phosphorylated is critical for SOCS1 binding and SOCS1-mediated degradation of ASK1. However, the kinase and phosphatase responsible for phosphorylation and dephosphorylation of ASK1 at Tyr-718 are unknown. In this study, we identified JAK2 and SHP2 as a Tyr-718-specific kinase and phosphatase, respectively. Interferon-γ (IFN-γ) induced degradation of ASK1 in normal but not in SOCS1-KO endothelial cells (EC). IFN-γ-induced tyrosine phosphorylation of ASK1 at Tyr-718 was blocked by a JAK2-specific inhibitor. IFN-γ enhanced the association between JAK2 and ASK1, and the ASK1-JAK2 complex was labile and was stabilized by the proteasomal inhibitor MG132. Furthermore, JAK2, but not JAK1, directly bound to and phosphorylated ASK1 at Tyr-718, leading to an enhanced association of ASK1 with SOCS1 and subsequent ASK1 degradation. Next, we showed that overexpression of the SH2-containing protein-tyrosine phosphatase-2 (SHP2) augmented, whereas a phosphatase-inactive mutant of SHP2 inhibited, TNF-induced ASK1 dephosphorylation. SHP2 associated with ASK1 in response to tumor necrosis factor in EC. An SHP-2 substrate-trapping mutant formed a complex with tyrosine-phosphorylated ASK1, suggesting that ASK1 is a direct SHP2 substrate. Moreover, SHP2 wild type, but not a catalytically inactive mutant, dissociated SOCS1 from ASK1. IFN-γ-induced ASK1 Tyr(P)-718 was enhanced in mouse EC deficient in SHP2 (SHP2-KO). In contrast, tumor necrosis factor-induced dephosphorylation of ASK1 at Tyr(P)-718 and activation of ASK1-JNK signaling, as well as EC apoptosis, are significantly reduced in SHP2-KO EC. Our data suggest that JAK2-SOCS1 and SHP2 reciprocally regulate ASK1 phosphorylation and stability in response to cytokines.Myocardial infarction due to atherosclerosis of coronary arteries remains the leading cause of death in the United States. It has become clear that increases in inflammatory mediators represent a common pathogenic mechanism for atherosclerosis (1). The vascular cell that normally limits the inflammatory and atherosclerotic process is the EC.³ Proinflammatory stimuli induce EC dysfunction, which is characterized by an enhanced sensitivity of vascular cells to proinflammatory and proapoptotic stimuli. Studies from our laboratory and others have demonstrated that ASK1 (apoptosis signal-regulating kinase-1), a member of MAP3K family (2, 3), is an effector of inflammation in EC (4–8). Almost all inflammatory stimuli such as tumor necrosis factor-α (TNF), interleukin-1 (IL-1), and reactive oxygen species activate ASK1. Activated ASK1 subsequently recruits and activates its downstream target MAP2Ks (MKK3/7 and MKK4/7), which in turn activate MAPKs (JNK and p38). Studies from ASK1-deficient mice have also linked ASK1 to cardiovascular pathogenesis. ASK1 deletion in mice attenuated angiotensin II-induced cardiac hypertrophy and remodeling. Neointimal formation due to proliferation of smooth muscle cells in a cuff injury model is also attenuated by ASK1 deletion in mice (9, 10).Although the linkage of ASK1 to inflammation is very strong, the mechanism by which inflammatory stimuli, including TNF, activate ASK1 is not fully understood. The identification of proteins associated with ASK1 and their regulation on ASK1 have provided some insights into the mechanism for ASK1 activation. ASK1 is a 170-kDa protein that is composed of an inhibitory N-terminal domain, an internal kinase domain, and a C-terminal regulatory domain. One important regulatory mechanism of ASK1 activity is its Ser/Thr phosphorylation and dephosphorylation by kinases and phosphatases. ASK1 is basally phosphorylated at Ser-967 by an unidentified kinase, and 14-3-3 binds to this site and inhibits ASK1 activity (11, 12). TNF activates ASK1 in part by dissociating these cellular inhibitors from ASK1 (4, 7). Recently, we have identified PP2A as a phosphatase in TNF-induced dephosphorylation of ASK1 Ser(P)-967 (13). In addition to the 14-3-3-binding site, Ser(P)-967, ASK1 is phosphorylated at Ser-83 by Akt, leading to inhibition of ASK1 activity. In contrast, autophosphorylation of ASK1 at Thr-838 leads to oligomerization and activation (14). Phosphorylation of Thr-845 can be negatively regulated by the phosphatase PP5 (15). Similarly, we found that the ASK1 autophosphorylation at Thr-813 and Thr-842 also positively regulates ASK1 signaling (16).In contrast to Ser/Thr phosphorylation, regulation of ASK1 by tyrosine phosphorylation is less well understood. We have recently shown that ASK1 is phosphorylated at Tyr-718, and this phosphorylation is critical for the binding to suppressor of cytokine signaling-1 (SOCS1), a subunit of ubiquitin ligase responsible for ASK1 degradation (17). Tyrosine phosphorylation of ASK1 is up-regulated in response to growth factors and cytokines such as IFN-γ, whereas this phosphorylation can be down-regulated by TNF treatment, resulting in ASK1 dissociation from SOCS1. However, the kinase and phosphatase responsible for phosphorylation and dephosphorylation of ASK1 at Tyr-718 are not known.The cytoplasmic tyrosine kinase, JAK2, autophosphorylates in response to growth factors and cytokines, including IFN-γ. JAK2 then activates cytokine receptors and other cytoplasmic proteins such as the STATs by phosphorylating their key tyrosine residue. The JAK/STAT pathway can be regulated by SH2-containing protein-tyrosine phosphatases such as SHP2 (18–20). SHP2 is ubiquitously expressed and composed of two SH2 domains on the N-terminal and C-terminal protein-tyrosine phosphatase (PTP) domain. The SH2 domain of SHP2 mediates the association with phosphotyrosine-containing proteins present on activated receptors as well as on activated JAKs and STATs; this association triggers activation of the tyrosine phosphatase domain and subsequent dephosphorylation of substrates. SHP2 signals downstream of receptor tyrosine kinases and cytokine receptors, and in most cases it serves to positively transduce signals from these receptors. In other instances SHP2 has been shown to exhibit inhibitory signaling properties by negatively regulating the JAK-STAT pathway (19).In this study, we demonstrate that the IFN-γ-activated kinase JAK2 and TNF-activated SHP2 are the tyrosine kinase and phosphatase for Tyr-718 on ASK1, respectively. The actions of both JAK2 and SHP2 affect protein turnover of ASK1 and thus regulate ASK1/JNK-dependent proinflammatory and proapoptotic pathways in EC.     

Protein Ionizable Groups: pK Values and Their Contribution to Protein Stability and Solubility

The structure, stability, solubility, and function of proteins depend on their net charge and on the ionization state of the individual residues. Consequently, biochemists are interested in the pK values of the ionizable groups in proteins and how these pK values depend on their environment. We review what has been learned about pK values of ionizable groups in proteins from experimental studies and discuss the important contributions they make to protein stability and solubility.     

Protein Kinase C and Extracellular Signal-Regulated Kinase Regulate Movement,Attachment, Pairing and Egg Release in Schistosoma mansoni

Protein kinases C (PKCs) and extracellular signal-regulated kinases (ERKs) are evolutionary conserved cell signalling enzymes that coordinate cell function. Here we have employed biochemical approaches using ‘smart’ antibodies and functional screening to unravel the importance of these enzymes to Schistosoma mansoni physiology. Various PKC and ERK isotypes were detected, and were differentially phosphorylated (activated) throughout the various S. mansoni life stages, suggesting isotype-specific roles and differences in signalling complexity during parasite development. Functional kinase mapping in adult worms revealed that activated PKC and ERK were particularly associated with the adult male tegument, musculature and oesophagus and occasionally with the oesophageal gland; other structures possessing detectable activated PKC and/or ERK included the Mehlis'' gland, ootype, lumen of the vitellaria, seminal receptacle and excretory ducts. Pharmacological modulation of PKC and ERK activity in adult worms using GF109203X, U0126, or PMA, resulted in significant physiological disturbance commensurate with these proteins occupying a central position in signalling pathways associated with schistosome muscular activity, neuromuscular coordination, reproductive function, attachment and pairing. Increased activation of ERK and PKC was also detected in worms following praziquantel treatment, with increased signalling associated with the tegument and excretory system and activated ERK localizing to previously unseen structures, including the cephalic ganglia. These findings support roles for PKC and ERK in S. mansoni homeostasis, and identify these kinase groups as potential targets for chemotherapeutic treatments against human schistosomiasis, a neglected tropical disease of enormous public health significance.     

Protein Kinase C-mediated Phosphorylation and Activation of PDE3A Regulate cAMP Levels in Human Platelets

The elevation of [cAMP]_i is an important mechanism of platelet inhibition and is regulated by the opposing activity of adenylyl cyclase and phosphodiesterase (PDE). In this study, we demonstrate that a variety of platelet agonists, including thrombin, significantly enhance the activity of PDE3A in a phosphorylation-dependent manner. Stimulation of platelets with the PAR-1 agonist SFLLRN resulted in rapid and transient phosphorylation of PDE3A on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹², in parallel with the PKC (protein kinase C) substrate, pleckstrin. Furthermore, phosphorylation and activation of PDE3A required the activation of PKC, but not of PI3K/PKB, mTOR/p70S6K, or ERK/RSK. Activation of PKC by phorbol esters also resulted in phosphorylation of the same PDE3A sites in a PKC-dependent, PKB-independent manner. This was further supported by the finding that IGF-1, which strongly activates PI3K/PKB, but not PKC, did not regulate PDE3A. Platelet activation also led to a PKC-dependent association between PDE3A and 14-3-3 proteins. In contrast, cAMP-elevating agents such as PGE₁ and forskolin-induced phosphorylation of Ser³¹² and increased PDE3A activity, but did not stimulate 14-3-3 binding. Finally, complete antagonism of PGE₁-evoked cAMP accumulation by thrombin required both G_i and PKC activation. Together, these results demonstrate that platelet activation stimulates PKC-dependent phosphorylation of PDE3A on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹² leading to a subsequent increase in cAMP hydrolysis and 14-3-3 binding.Upon vascular injury, platelets adhere to the newly exposed subintimal collagen and undergo activation leading to platelet spreading to cover the damaged region and release of thrombogenic factors such as ADP and thromboxane A₂. In addition, platelets are activated by thrombin, which is generated as a result of activation of the coagulation pathway, and stimulates platelets by cleaving the protease-activated receptors (PAR),² PAR-1 and PAR-4. The final common pathway is the exposure of fibrinogen binding sites on integrin α_IIbβ₃ resulting in platelet aggregation and thrombus formation.Thrombin-mediated cleavage of PARs leads to activation of phospholipase C β (PLC), hydrolysis of phosphatidylinositol (PI) 4,5-bisphosphate and a subsequent increase in [Ca²⁺]_i and activation of protein kinase C (PKC). Protein kinase C contributes to platelet activation both directly, through affinity regulation of the fibrinogen receptor, integrin α_IIbβ₃ (1), and indirectly by enhancing degranulation (2). Thrombin also stimulates activation of PI 3-kinases and subsequent generation of PI (3, 4, 5) trisphosphate and PI (3, 4) bisphosphate (3), which recruit protein kinase B (PKB) to the plasma membrane where it becomes phosphorylated and activated.Platelet activation is opposed by agents that raise intracellular 3′-5′-cyclic adenosine monophosphate ([cAMP]_i). cAMP is a powerful inhibitory second messenger that down-regulates platelet function by interfering with Ca²⁺ homeostasis, degranulation and integrin activation (4). Synthesis of cAMP is stimulated by mediators such as prostaglandin I₂ (PGI₂), which bind to G_s-coupled receptors leading to activation of adenylate cyclase (AC). This inhibitory pathway is opposed by thrombin, which inhibits the elevation of cAMP indirectly via autocrine activation of the G_i-coupled ADP receptor P2Y₁₂. cAMP signaling is terminated by hydrolysis to biologically inert 5′-AMP by 3′-phosphodiesterases. Platelets express two cAMP phosphodiesterase isoforms, cGMP-stimulated PDE2 and cGMP-inhibited PDE3A. PDE3A is the most abundant isoform in platelets and has a ∼250-fold lower K_m for cAMP than PDE2 (4). As a consequence of these properties, PDE3A exerts a greater influence on cAMP homeostasis, particularly at resting levels. The importance of PDE3A in platelet function is further emphasized by the finding that the PDE3A inhibitors cilostamide and milrinone raise basal cAMP levels and strongly inhibit thrombin-induced platelet activation (5). Furthermore, PDE3A^-/- mice demonstrate increased resting levels of platelet cAMP and are protected against a model of pulmonary thrombosis (6). In contrast, the PDE2 inhibitor EHNA has no significant effect on cAMP levels and platelet aggregation (7, 8). The activity of PDE3A is therefore essential to maintain low equilibrium levels of cAMP and determine the threshold for platelet activation (7).Like its paralogue PDE3B, it has recently become clear that PDE3A activity can be positively regulated by phosphorylation in platelets and human oocytes (9, 10). There is some evidence that PKB may be involved in this regulation, although the phosphorylation sites are poorly characterized. In contrast, phosphorylation of PDE3A in HeLa cells was stimulated by phorbol esters and blocked by inhibitors of PKC (11). In this study, we aimed to identify the signaling pathways and phosphorylation sites that are involved in regulation of platelet PDE3A. Here, we show strong evidence that PKC, and not PKB, is involved in agonist-stimulated PDE3A phosphorylation on Ser³¹², Ser⁴²⁸, Ser⁴³⁸, Ser⁴⁶⁵, and Ser⁴⁹², leading to an increase in PDE3A activity, 14-3-3 binding and modulation of intracellular cAMP levels.     

干扰素诱导蛋白p204调节心肌分化的机制及应用前景

干扰素诱导蛋白p200家族蛋白包括6种鼠类及4种人类家族成员,具有共同的特征结构,广泛参与调节细胞增殖和分化、衰老和凋亡,在自身免疫反应、抗病毒及抗癌等领域发挥着重要的作用。内源性的p200家族蛋白鼠p204在心肌及骨骼肌表达最高,提示其在肌分化中起着重要作用。本文联系p204的分子结构及调节细胞生长与分化的功能,阐述p204促骨骼肌成肌细胞及胚胎癌细胞分化的机制,及对心肌损伤后心肌再生的应用前景。     

Subunits of the Drosophila Actin-Capping Protein Heterodimer Regulate Each Other at Multiple Levels

The actin-Capping Protein heterodimer, composed of the α and β subunits, is a master F-actin regulator. In addition to its role in many cellular processes, Capping Protein acts as a main tumor suppressor module in Drosophila and in humans, in part, by restricting the activity of Yorkie/YAP/TAZ oncogenes. We aimed in this report to understand how both subunits regulate each other in vivo. We show that the levels and capping activities of both subunits must be tightly regulated to control F-actin levels and consequently growth of the Drosophila wing. Overexpressing capping protein α and β decreases both F-actin levels and tissue growth, while expressing forms of Capping Protein that have dominant negative effects on F-actin promote tissue growth. Both subunits regulate each other''s protein levels. In addition, overexpressing one of the subunit in tissues knocked-down for the other increases the mRNA and protein levels of the subunit knocked-down and compensates for its loss. We propose that the ability of the α and β subunits to control each other''s levels assures that a pool of functional heterodimer is produced in sufficient quantities to restrict the development of tumor but not in excess to sustain normal tissue growth.     

Streptococcus pneumoniae Translocates into the Myocardium and Forms Unique Microlesions That Disrupt Cardiac Function

Hospitalization of the elderly for invasive pneumococcal disease is frequently accompanied by the occurrence of an adverse cardiac event; these are primarily new or worsened heart failure and cardiac arrhythmia. Herein, we describe previously unrecognized microscopic lesions (microlesions) formed within the myocardium of mice, rhesus macaques, and humans during bacteremic Streptococcus pneumoniae infection. In mice, invasive pneumococcal disease (IPD) severity correlated with levels of serum troponin, a marker for cardiac damage, the development of aberrant cardiac electrophysiology, and the number and size of cardiac microlesions. Microlesions were prominent in the ventricles, vacuolar in appearance with extracellular pneumococci, and remarkable due to the absence of infiltrating immune cells. The pore-forming toxin pneumolysin was required for microlesion formation but Interleukin-1β was not detected at the microlesion site ruling out pneumolysin-mediated pyroptosis as a cause of cell death. Antibiotic treatment resulted in maturing of the lesions over one week with robust immune cell infiltration and collagen deposition suggestive of long-term cardiac scarring. Bacterial translocation into the heart tissue required the pneumococcal adhesin CbpA and the host ligands Laminin receptor (LR) and Platelet-activating factor receptor. Immunization of mice with a fusion construct of CbpA or the LR binding domain of CbpA with the pneumolysin toxoid L460D protected against microlesion formation. We conclude that microlesion formation may contribute to the acute and long-term adverse cardiac events seen in humans with IPD.     

Fbw7 and Usp28 Regulate Myc Protein Stability in Response to DNA Damage

The cellular levels of the Myc oncoprotein are critical determinants of cell proliferation, cell growth and apoptosis and are tightly regulated by external growth factors. Levels of Myc oncoprotein also decline in response to intracellular stress signals such as DNA damage. We show here that this decline is in part due to proteasomal degradation and that it is mediated by the Fbw7 ubiquitin ligase. We have shown previously that the ubiquitin-specific protease Usp28, binds to the nucleoplasmic isoform of Fbw7, Fbw7α, and counteracts its function in mammalian cells. Usp28 dissociates from Fbw7α in response to UV irradiation, providing a mechanism how Fbw7-mediated degradation of Myc is enhanced upon DNA damage. Our data extend previous observations that link Myc function to the cellular response to DNA damage.     

Netrins and Wnts Function Redundantly to Regulate Antero-Posterior and Dorso-Ventral Guidance in C. elegans

Guided migrations of cells and developing axons along the dorso-ventral (D/V) and antero-posterior (A/P) body axes govern tissue patterning and neuronal connections. In C. elegans, as in vertebrates, D/V and A/P graded distributions of UNC-6/Netrin and Wnts, respectively, provide instructive polarity information to guide cells and axons migrating along these axes. By means of a comprehensive genetic analysis, we found that simultaneous loss of Wnt and Netrin signaling components reveals previously unknown and unexpected redundant roles for Wnt and Netrin signaling pathways in both D/V and A/P guidance of migrating cells and axons in C. elegans, as well as in processes essential for organ function and viability. Thus, in addition to providing polarity information for migration along the axis of their gradation, Wnts and Netrin are each able to guide migrations orthogonal to the axis of their gradation. Netrin signaling not only functions redundantly with some Wnts, but also counterbalances the effects of others to guide A/P migrations, while the involvement of Wnt signaling in D/V guidance identifies Wnt signaling as one of the long sought mechanisms that functions in parallel to Netrin signaling to promote D/V guidance of cells and axons. These findings provide new avenues for deciphering how A/P and D/V guidance signals are integrated within the cell to establish polarity in multiple biological processes, and implicate broader roles for Netrin and Wnt signaling - roles that are currently masked due to prevalent redundancy.     

Dopamine Receptors Antagonistically Regulate Behavioral Choice between Conflicting Alternatives in C. elegans

Caenorhabditis elegans is a useful model to study the neuronal or molecular basis for behavioral choice, a specific form of decision-making. Although it has been implied that both D1-like and D2-like dopamine receptors may contribute to the control of decision-making in mammals, the genetic interactions between D1-like and D2-like dopamine receptors in regulating decision-making are still largely unclear. In the present study, we investigated the molecular control of behavioral choice between conflicting alternatives (diacetyl and Cu²⁺) by D1-like and D2-like dopamine receptors and their possible genetic interactions with C. elegans as the assay system. In the behavioral choice assay system, mutation of dop-1 gene encoding D1-like dopamine receptor resulted in the enhanced tendency to cross the Cu²⁺ barrier compared with wild-type. In contrast, mutations of dop-2 or dop-3 gene encoding D2-like dopamine receptor caused the weak tendency to cross the Cu²⁺ barrier compared with wild-type. During the control of behavioral choice, DOP-3 antagonistically regulated the function of DOP-1. The behavioral choice phenotype of dop-2; dop-1dop-3 triple mutant further confirmed the possible antagonistic function of D2-like dopamine receptor on D1-like dopamine receptor in regulating behavioral choice. The genetic assays further demonstrate that DOP-3 might act through Gα_o signaling pathway encoded by GOA-1 and EGL-10, and DOP-1 might act through Gα_q signaling pathway encoded by EGL-30 and EAT-16 to regulate the behavioral choice. DOP-1 might function in cholinergic neurons to regulate the behavioral choice, whereas DOP-3 might function in GABAergic neurons, RIC, and SIA neurons to regulate the behavioral choice. In this study, we provide the genetic evidence to indicate the antagonistic relationship between D1-like dopamine receptor and D2-like dopamine receptor in regulating the decision-making of animals. Our data will be useful for understanding the complex functions of dopamine receptors in regulating decision-making in animals.