Tropomyosin Dephosphorylation Results in Compensated Cardiac Hypertrophy

Phosphorylation of tropomyosin (Tm) has been shown to vary in mouse models of cardiac hypertrophy. Little is known about the in vivo role of Tm phosphorylation. This study examines the consequences of Tm dephosphorylation in the murine heart. Transgenic (TG) mice were generated with cardiac specific expression of α-Tm with serine 283, the phosphorylation site of Tm, mutated to alanine. Echocardiographic analysis and cardiomyocyte cross-sectional area measurements show that α-Tm S283A TG mice exhibit a hypertrophic phenotype at basal levels. Interestingly, there are no alterations in cardiac function, myofilament calcium (Ca²⁺) sensitivity, cooperativity, or response to β-adrenergic stimulus. Studies of Ca²⁺ handling proteins show significant increases in sarcoplasmic reticulum ATPase (SERCA2a) protein expression and an increase in phospholamban phosphorylation at serine 16, similar to hearts under exercise training. Compared with controls, the decrease in phosphorylation of α-Tm results in greater functional defects in TG animals stressed by transaortic constriction to induce pressure overload-hypertrophy. This is the first study to investigate the in vivo role of Tm dephosphorylation under both normal and cardiac stress conditions, documenting a role for Tm dephosphorylation in the maintenance of a compensated or physiological phenotype. Collectively, these results suggest that modification of the Tm phosphorylation status in the heart, depending upon the cardiac state/condition, may modulate the development of cardiac hypertrophy.     

Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand Specificity

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits Shp2 phosphatase activity. We identified two key amino acids in Shp2 that are phosphorylated by PKA. Thr-73 contributes a helix cap to helix αB within the N-terminal SH2 domain of Shp2, whereas Ser-189 occupies an equivalent position within the C-terminal SH2 domain. Utilizing double mutant PKA phosphodeficient (T73A/S189A) and phosphomimetic (T73D/S189D) constructs, in vitro binding assays, and phosphatase activity assays, we demonstrate that phosphorylation of these residues disrupts Shp2 interaction with tyrosine-phosphorylated ligands and inhibits its protein-tyrosine phosphatase activity. Overall, our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Therefore, although induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote this compensatory response.     

Src Homology 2 Domain-containing Phosphatase 2 (Shp2) Is a Component of the A-kinase-anchoring Protein (AKAP)-Lbc Complex and Is Inhibited by Protein Kinase A (PKA) under Pathological Hypertrophic Conditions in the Heart

Pathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Our results identify a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that the tyrosine phosphatase, Shp2, is a component of the A-kinase-anchoring protein (AKAP)-Lbc complex. AKAP-Lbc facilitates PKA phosphorylation of Shp2, which inhibits its protein-tyrosine phosphatase activity. Given the important cardiac roles of both AKAP-Lbc and Shp2, we investigated the AKAP-Lbc-Shp2 interaction in the heart. AKAP-Lbc-tethered PKA is implicated in cardiac hypertrophic signaling; however, mechanism of PKA action is unknown. Mutations resulting in loss of Shp2 catalytic activity are also associated with cardiac hypertrophy and congenital heart defects. Our data indicate that AKAP-Lbc integrates PKA and Shp2 signaling in the heart and that AKAP-Lbc-associated Shp2 activity is reduced in hypertrophic hearts in response to chronic β-adrenergic stimulation and PKA activation. Thus, while induction of cardiac hypertrophy is a multifaceted process, inhibition of Shp2 activity through AKAP-Lbc-anchored PKA is a previously unrecognized mechanism that may promote compensatory cardiac hypertrophy.     

Short Forms of Ste20-related Proline/Alanine-rich Kinase (SPAK) in the Kidney Are Created by Aspartyl Aminopeptidase (Dnpep)-mediated Proteolytic Cleavage

The Ste20-related kinase SPAK regulates sodium, potassium, and chloride transport in a variety of tissues. Recently, SPAK fragments, which lack the catalytic domain and are inhibitory to Na⁺ transporters, have been detected in kidney. It has been hypothesized that the fragments originate from alternative translation start sites, but their precise origin is unknown. Here, we demonstrate that kidney lysate possesses proteolytic cleavage activity toward SPAK. Ion exchange and size exclusion chromatography combined with mass spectrometry identified the protease as aspartyl aminopeptidase. The presence of the protease was verified in the active fractions, and recombinant aspartyl aminopeptidase recapitulated the cleavage pattern observed with kidney lysate. Identification of the sites of cleavage by mass spectrometry allowed us to test the function of the smaller fragments and demonstrate their inhibitory action toward the Na⁺-K⁺-2Cl⁻ cotransporter, NKCC2.     

Dopamine- and cAMP-regulated Phosphoprotein of 32-kDa (DARPP-32)-dependent Activation of Extracellular Signal-regulated Kinase (ERK) and Mammalian Target of Rapamycin Complex 1 (mTORC1) Signaling in Experimental Parkinsonism

Dyskinesia, a motor complication caused by prolonged administration of the antiparkinsonian drug l-3,4-dihydroxyphenylalanine (l-DOPA), is accompanied by activation of cAMP signaling and hyperphosphorylation of the dopamine- and cAMP-regulated phosphoprotein of 32 kDa (DARPP-32). Here, we show that the abnormal phosphorylation of DARPP-32 occurs specifically in medium spiny neurons (MSNs) expressing dopamine D1 receptors (D1R). Using mice in which DARPP-32 is selectively deleted in D1R-expressing MSNs, we demonstrate that this protein is required for l-DOPA-induced activation of the extracellular signal-regulated protein kinases 1 and 2 and the mammalian target of rapamycin complex 1 (mTORC1) pathways, which are implicated in dyskinesia. We also show that mutation of the phosphorylation site for cAMP-dependent protein kinase on DARPP-32 attenuates l-DOPA-induced dyskinesia and reduces the concomitant activations of ERK and mTORC1 signaling. These studies demonstrate that, in D1R-expressing MSNs, l-DOPA-induced activation of ERK and mTORC1 requires DARPP-32 and indicates the importance of the cAMP/DARPP-32 signaling cascade in dyskinesia.     

Exogenous NAD Blocks Cardiac Hypertrophic Response via Activation of the SIRT3-LKB1-AMP-activated Kinase Pathway

Since the discovery of NAD-dependent deacetylases, sirtuins, it has been recognized that maintaining intracellular levels of NAD is crucial for the management of stress response of cells. Here we show that agonist-induced cardiac hypertrophy is associated with loss of intracellular levels of NAD, but not exercise-induced physiologic hypertrophy. Exogenous addition of NAD was capable of maintaining intracellular levels of NAD and blocking the agonist-induced cardiac hypertrophic response in vitro as well as in vivo. NAD treatment blocked the activation of pro-hypertrophic Akt1 signaling, and augmented the activity of anti-hypertrophic LKB1-AMPK signaling in the heart, which prevented subsequent induction of mTOR-mediated protein synthesis. By using gene knock-out and transgenic mouse models of SIRT3 and SIRT1, we showed that the anti-hypertrophic effects of exogenous NAD are mediated through activation of SIRT3, but not SIRT1. SIRT3 deacetylates and activates LKB1, thus augmenting the activity of the LKB1-AMPK pathway. These results reveal a novel role of NAD as an inhibitor of cardiac hypertrophic signaling, and suggest that prevention of NAD depletion may be critical in the treatment of cardiac hypertrophy and heart failure.     

The c-Jun N-terminal Kinase (JNK)-binding Protein (JNKBP1) Acts as a Negative Regulator of NOD2 Protein Signaling by Inhibiting Its Oligomerization Process

NOD2 is one of the best characterized members of the cytosolic NOD-like receptor family. NOD2 is able to sense muramyl dipeptide, a specific bacterial cell wall component, and to subsequently induce various signaling pathways leading to NF-κB activation and autophagy, both events contributing to an efficient innate and adaptive immune response. Interestingly, loss-of-function NOD2 variants were associated with a higher susceptibility for Crohn disease, which highlights the physiological importance of proper regulation of NOD2 activity. We performed a biochemical screen to search for new NOD2 regulators. We identified a new NOD2 partner, c-Jun N-terminal kinase-binding protein 1 (JNKBP1), a scaffold protein characterized by an N-terminal WD-40 domain. JNKBP1, through its WD-40 domain, binds to NOD2 following muramyl dipeptide activation. This interaction attenuates NOD2-mediated NF-κB activation and IL-8 secretion as well as NOD2 antibacterial activity. JNKBP1 exerts its repressor effect by disturbing NOD2 oligomerization and RIP2 tyrosine phosphorylation, both steps required for downstream NOD2 signaling. We furthermore showed that JNKBP1 and NOD2 are co-expressed in the human intestinal epithelium and in immune cells recruited in the lamina propria, which suggests that JNKBP1 contributes to maintain NOD2-mediated intestinal immune homeostasis.     

Long Term Ablation of Protein Kinase A (PKA)-mediated Cardiac Troponin I Phosphorylation Leads to Excitation-Contraction Uncoupling and Diastolic Dysfunction in a Knock-in Mouse Model of Hypertrophic Cardiomyopathy

The cardiac troponin I (cTnI) R21C (cTnI-R21C) mutation has been linked to hypertrophic cardiomyopathy and renders cTnI incapable of phosphorylation by PKA in vivo. Echocardiographic imaging of homozygous knock-in mice expressing the cTnI-R21C mutation shows that they develop hypertrophy after 12 months of age and have abnormal diastolic function that is characterized by longer filling times and impaired relaxation. Electrocardiographic analyses show that older R21C mice have elevated heart rates and reduced cardiovagal tone. Cardiac myocytes isolated from older R21C mice demonstrate that in the presence of isoproterenol, significant delays in Ca²⁺ decay and sarcomere relaxation occur that are not present at 6 months of age. Although isoproterenol and stepwise increases in stimulation frequency accelerate Ca²⁺-transient and sarcomere shortening kinetics in R21C myocytes from older mice, they are unable to attain the corresponding WT values. When R21C myocytes from older mice are treated with isoproterenol, evidence of excitation-contraction uncoupling is indicated by an elevation in diastolic calcium that is frequency-dissociated and not coupled to shorter diastolic sarcomere lengths. Myocytes from older mice have smaller Ca²⁺ transient amplitudes (2.3-fold) that are associated with reductions (2.9-fold) in sarcoplasmic reticulum Ca²⁺ content. This abnormal Ca²⁺ handling within the cell may be attributed to a reduction (2.4-fold) in calsequestrin expression in conjunction with an up-regulation (1.5-fold) of Na⁺-Ca²⁺ exchanger. Incubation of permeabilized cardiac fibers from R21C mice with PKA confirmed that the mutation prevents facilitation of mechanical relaxation. Altogether, these results indicate that the inability to enhance myofilament relaxation through cTnI phosphorylation predisposes the heart to abnormal diastolic function, reduced accessibility of cardiac reserves, dysautonomia, and hypertrophy.     

Prostate-derived Sterile 20-like Kinases (PSKs/TAOKs) Phosphorylate Tau Protein and Are Activated in Tangle-bearing Neurons in Alzheimer Disease

In Alzheimer disease (AD), the microtubule-associated protein tau is highly phosphorylated and aggregates into characteristic neurofibrillary tangles. Prostate-derived sterile 20-like kinases (PSKs/TAOKs) 1 and 2, members of the sterile 20 family of kinases, have been shown to regulate microtubule stability and organization. Here we show that tau is a good substrate for PSK1 and PSK2 phosphorylation with mass spectrometric analysis of phosphorylated tau revealing more than 40 tau residues as targets of these kinases. Notably, phosphorylated residues include motifs located within the microtubule-binding repeat domain on tau (Ser-262, Ser-324, and Ser-356), sites that are known to regulate tau-microtubule interactions. PSK catalytic activity is enhanced in the entorhinal cortex and hippocampus, areas of the brain that are most susceptible to Alzheimer pathology, in comparison with the cerebellum, which is relatively spared. Activated PSK is associated with neurofibrillary tangles, dystrophic neurites surrounding neuritic plaques, neuropil threads, and granulovacuolar degeneration bodies in AD brain. By contrast, activated PSKs and phosphorylated tau are rarely detectible in immunostained control human brain. Our results demonstrate that tau is a substrate for PSK and suggest that this family of kinases could contribute to the development of AD pathology and dementia.     

Dual Function of UNC-51-like Kinase 3 (Ulk3) in the Sonic Hedgehog Signaling Pathway

The Sonic hedgehog (Shh) signaling pathway controls a variety of developmental processes and is implicated in tissue homeostasis maintenance and neurogenesis in adults. Recently, we identified Ulk3 as an active kinase able to positively regulate Gli proteins, mediators of the Shh signaling in mammals. Here, we provide several lines of evidence that Ulk3 participates in the transduction of the Shh signal also independently of its kinase activity. We demonstrate that Ulk3 through its kinase domain interacts with Suppressor of Fused (Sufu), a protein required for negative regulation of Gli proteins. Sufu blocks Ulk3 autophosphorylation and abolishes its ability to phosphorylate and positively regulate Gli proteins. We show that Shh signaling destabilizes the Sufu-Ulk3 complex and induces the release of Ulk3. We demonstrate that the Sufu-Ulk3 complex, when co-expressed with Gli2, promotes generation of the Gli2 repressor form, and that reduction of the Ulk3 mRNA level in Shh-responsive cells results in higher potency of the cells to transmit the Shh signal. Our data suggests a dual function of Ulk3 in the Shh signal transduction pathway and propose an additional way of regulating Gli proteins by Sufu, through binding to and suppression of Ulk3.     

Opposing actions of extracellular signal-regulated kinase (ERK) and signal transducer and activator of transcription 3 (STAT3) in regulating microtubule stabilization during cardiac hypertrophy

Excessive proliferation and stabilization of the microtubule (MT) array in cardiac myocytes can accompany pathological cardiac hypertrophy, but the molecular control of these changes remains poorly characterized. In this study, we examined MT stabilization in two independent murine models of heart failure and revealed increases in the levels of post-translationally modified stable MTs, which were closely associated with STAT3 activation. To explore the molecular signaling events contributing to control of the cardiac MT network, we stimulated cardiac myocytes with an α-adrenergic agonist phenylephrine (PE), and observed increased tubulin content without changes in detyrosinated (glu-tubulin) stable MTs. In contrast, the hypertrophic interleukin-6 (IL6) family cytokines increased both the glu-tubulin content and glu-MT density. When we examined a role for ERK in regulating cardiac MTs, we showed that the MEK/ERK-inhibitor U0126 increased glu-MT density in either control cardiac myocytes or following exposure to hypertrophic agents. Conversely, expression of an activated MEK1 mutant reduced glu-tubulin levels. Thus, ERK signaling antagonizes stabilization of the cardiac MT array. In contrast, inhibiting either JAK2 with AG490, or STAT3 signaling with Stattic or siRNA knockdown, blocked cytokine-stimulated increases in glu-MT density. Furthermore, the expression of a constitutively active STAT3 mutant triggered increased glu-MT density in the absence of hypertrophic stimulation. Thus, STAT3 activation contributes substantially to cytokine-stimulated glu-MT changes. Taken together, our results highlight the opposing actions of STAT3 and ERK pathways in the regulation of MT changes associated with cardiac myocyte hypertrophy.     

Distinct Sarcomeric Substrates Are Responsible for Protein Kinase D-mediated Regulation of Cardiac Myofilament Ca2+ Sensitivity and Cross-bridge Cycling

Protein kinase D (PKD), a serine/threonine kinase with emerging cardiovascular functions, phosphorylates cardiac troponin I (cTnI) at Ser²²/Ser²³, reduces myofilament Ca²⁺ sensitivity, and accelerates cross-bridge cycle kinetics. Whether PKD regulates cardiac myofilament function entirely through cTnI phosphorylation at Ser²²/Ser²³ remains to be established. To determine the role of cTnI phosphorylation at Ser²²/Ser²³ in PKD-mediated regulation of cardiac myofilament function, we used transgenic mice that express cTnI in which Ser²²/Ser²³ are substituted by nonphosphorylatable Ala (cTnI-Ala₂). In skinned myocardium from wild-type (WT) mice, PKD increased cTnI phosphorylation at Ser²²/Ser²³ and decreased the Ca²⁺ sensitivity of force. In contrast, PKD had no effect on the Ca²⁺ sensitivity of force in myocardium from cTnI-Ala₂ mice, in which Ser²²/Ser²³ were unavailable for phosphorylation. Surprisingly, PKD accelerated cross-bridge cycle kinetics similarly in myocardium from WT and cTnI-Ala₂ mice. Because cardiac myosin-binding protein C (cMyBP-C) phosphorylation underlies cAMP-dependent protein kinase (PKA)-mediated acceleration of cross-bridge cycle kinetics, we explored whether PKD phosphorylates cMyBP-C at its PKA sites, using recombinant C1C2 fragments with or without site-specific Ser/Ala substitutions. Kinase assays confirmed that PKA phosphorylates Ser²⁷³, Ser²⁸², and Ser³⁰², and revealed that PKD phosphorylates only Ser³⁰². Furthermore, PKD phosphorylated Ser³⁰² selectively and to a similar extent in native cMyBP-C of skinned myocardium from WT and cTnI-Ala₂ mice, and this phosphorylation occurred throughout the C-zones of sarcomeric A-bands. In conclusion, PKD reduces myofilament Ca²⁺ sensitivity through cTnI phosphorylation at Ser²²/Ser²³ but accelerates cross-bridge cycle kinetics by a distinct mechanism. PKD phosphorylates cMyBP-C at Ser³⁰², which may mediate the latter effect.     

Microtubule-associated Protein/Microtubule Affinity-regulating Kinase 4 (MARK4) Is a Negative Regulator of the Mammalian Target of Rapamycin Complex 1 (mTORC1)

The mammalian target of rapamycin (mTOR) is a central cell growth regulator. It resides in two protein complexes, which in mammals are referred to as mTORC1 and mTORC2. mTORC1, which is directly inhibited by rapamycin, promotes cell growth by stimulating protein synthesis and inhibiting autophagy. A wide range of extra and intracellular signals, including growth factors, nutrients, energy levels, and various stress conditions, regulates mTORC1. Dysregulation of mTORC1 contributes to many human diseases, including cancer, cardiovascular disease, autoimmunity, and metabolic disorder. In this study, we identified MARK4, an AMP-activated kinase-related kinase, as a negative regulator of mTORC1. In Drosophila S2 cells and mammalian cells, knockdown of MARK family member increased mTORC1 activity, whereas overexpression of MARK4 in mammalian cells significantly inhibited mTORC1 activity. Interestingly, MARK4 selectively inhibits mTORC1 activation by Rag GTPases, which are involved in amino acid signaling, but does not inhibit the effect of Rheb, which directly binds to and activates mTORC1. In addition, we found that MARK4 phosphorylates Raptor, a key component of mTORC1, and this phosphorylation may interfere with Raptor-Rag interaction. Our data demonstrate MARK4 as a new negative regulator of mTORC1.     

Inhibition of G-protein-coupled Receptor Kinase 2 (GRK2) Triggers the Growth-promoting Mitogen-activated Protein Kinase (MAPK) Pathway

Inhibition of G-protein-coupled receptor kinase 2 (GRK2) is an emerging treatment option for heart failure. Because GRK2 is also indispensable for growth and development, we analyzed the impact of GRK2 inhibition on cell growth and proliferation. Inhibition of GRK2 by the dominant-negative GRK2-K220R did not affect the proliferation of cultured cells. In contrast, upon xenograft transplantation of cells into immunodeficient mice, the dominant-negative GRK2-K220R or a GRK2-specific peptide inhibitor increased tumor mass. The enhanced tumor growth upon GRK2 inhibition was attributed to the growth-promoting MAPK pathway because dual inhibition of the GRK2 and RAF-MAPK axis by the Raf kinase inhibitor protein (RKIP) did not increase tumor mass. The MAPK cascade contributed to the cardioprotective profile of GRK2 inhibition by preventing cardiomyocyte death, whereas dual inhibition of RAF/MAPK and GRK2 by RKIP induced cardiomyocyte apoptosis, cardiac dysfunction, and signs of heart failure. Thus, cardioprotective signaling induced by GRK2 inhibition is overlapping with tumor growth promotion.     

Phospholipase C epsilon scaffolds to muscle-specific A kinase anchoring protein (mAKAPbeta) and integrates multiple hypertrophic stimuli in cardiac myocytes

To define a role for phospholipase Cε (PLCε) signaling in cardiac myocyte hypertrophic growth, PLCε protein was depleted from neonatal rat ventricular myocytes (NRVMs) using siRNA. NRVMs with PLCε depletion were stimulated with endothelin (ET-1), norepinephrine, insulin-like growth factor-1 (IGF-1), or isoproterenol and assessed for development of hypertrophy. PLCε depletion dramatically reduced hypertrophic growth and gene expression induced by all agonists tested. PLCε catalytic activity was required for hypertrophy development, yet PLCε depletion did not reduce global agonist-stimulated inositol phosphate production, suggesting a requirement for localized PLC activity. PLCε was found to be scaffolded to a muscle-specific A kinase anchoring protein (mAKAPβ) in heart and NRVMs, and mAKAPβ localizes to the nuclear envelope in NRVMs. PLCε-mAKAP interaction domains were defined and overexpressed to disrupt endogenous mAKAPβ-PLCε complexes in NRVMs, resulting in significantly reduced ET-1-dependent NRVM hypertrophy. We propose that PLCε integrates multiple upstream signaling pathways to generate local signals at the nucleus that regulate hypertrophy.     

Hsp90 Interaction with INrf2(Keap1) Mediates Stress-induced Nrf2 Activation

INrf2(Keap1) functions as an adapter for Cul3/Rbx1-mediated degradation of Nrf2. In response to stress, Nrf2 is released from INrf2 and translocates inside the nucleus leading to activation of cytoprotective proteins critical in protection against adverse effects including cancer. We demonstrate here a novel role of heat shock protein 90 (Hsp90) in control of the INrf2 and Nrf2 activation. Hsp90 interacted with INrf2 that leds to stabilization of INrf2 during heat shock stress. Domain mapping showed the requirement of INrf2-NTR and the Hsp90-CLD region for interaction of Hsp90 with INrf2. Heat shock and antioxidants induced Hsp90, and casein kinase 2 (CK2) phosphorylated INrf2Thr55. This led to increased Hsp90-INrf2 interaction, dissociation of the Rbx1/Cul3·INrf2·Nrf2 complex, and activation of Nrf2. Inhibitors of CK2 and Hsp90, and mutation of INrf2Thr55 abolished the Hsp90-INrf2 interaction and downstream signaling. INrf2 is released from Hsp90 once the heat shock or antioxidant stress subsidized, thereby allowing INrf2 to interact with Nrf2 and facilitate Nrf2 ubiquitination and degradation. The results together demonstrate a novel role for the stress-induced Hsp90-INrf2 interaction in regulation of Nrf2 activation and induction of cytoprotective proteins.