Redox and proteolytic regulation of cardiomyocyte ß1-adrenergic receptors - a novel paradigm for the regulation of catecholamine responsiveness in the heart.

Conventional models view ß1-adrenergic receptors (ß1ARs) as full-length proteins that activate signaling pathways that influence contractile function and ventricular remodeling - and are susceptible to agonist-dependent desensitization. This perspective summarizes recent studies from my laboratory showing that post-translational processing of the ß1-adrenergic receptor N-terminus results in the accumulation of both full-length and N-terminally truncated forms of the ß1AR that differ in their signaling properties. We also implicate oxidative stress and ß1AR cleavage by elastase as two novel mechanisms that would (in the setting of cardiac injury or inflammation) lead to altered or decreased ß1AR responsiveness.

Beta1-Adrenergic Receptor Cleavage and Regulation by Elastase.

The decrease in ß1-adrenergic receptor responsiveness in heart failure is attributed conventionally to agonist-dependent desensitization. We identify elastase-dependent ß1-adrenergic receptor cleavage as a novel proteolytic mechanism that disrupts ß1-adrenergic receptor responsiveness in the setting of tissue injury or inflammation.

Lipid-independent activation of a muscle-specific PKCα splicing variant.

Protein kinase C-α (PKCα) plays a major role in a diverse range of cellular processes. Studies to date have defined the regulatory controls and function of PKCα entirely based upon the previously annotated ubiquitously expressed prototypical isoform. From RNA-seq-based transcriptome analysis in murine heart, we identified a previously unannotated PKCα variant produced by alternative RNA splicing. This PKCα transcript variant, which we named PKCα-novel exon (PKCα-NE), contains an extra exon between exon 16 and exon 17, and is specifically detected in adult mouse cardiac and skeletal muscle, but not other tissues; it is also detected in human hearts. This transcript variant yields a PKCα isoform with additional 16 amino acids inserted in its COOH-terminal variable region. Although the canonical PKCα enzyme is a lipid-dependent kinase, in vitro kinase assays show that PKCα-NE displays a high level of basal lipid-independent catalytic activity. Our unbiased proteomic analysis identified a specific interaction between PKCα-NE and eukaryotic elongation factor-1α (eEF1A1). Studies in cardiomyocytes link PKCα-NE expression to an increase in eEF1A1 phosphorylation and elevated protein synthesis. In summary, we have identified a previously uncharacterized muscle-specific PKCα splicing variant, PKCα-NE, with distinct biochemical properties that plays a unique role in the control of the protein synthesis machinery in cardiomyocytes.NEW & NOTEWORTHY PKCα is an important signaling molecule extensively studied in many cellular processes. However, no isoforms have been reported for PKCα except one prototypic isoform. Alternative mRNA splicing of Prkca gene was detected for the first time in rodent and human cardiac tissue, which can produce a previously unknown PKCα-novel exon (NE) isoform. The biochemistry and molecular effects of PKCα-NE are markedly different from PKCα wild type, suggesting potential functional diversity of PKCα signaling in muscle.

Trypsin cleavage of the ß1-adrenergic receptor.

ß1-Adrenergic receptors (ß1ARs) are the principal mediators of catecholamine action in cardiomyocytes. We previously showed that ß1ARs accumulate as both full-length and NH2-terminally truncated species in cells, that maturational processing of full-length ß1ARs to an NH2-terminally truncated form is attributable to O-glycan-regulated proteolytic cleavage of the ß1AR NH2-terminus at R31 ↓ L32 by ADAM17, and that NH2-terminally truncated ß1ARs remain signaling competent but they acquire a distinct signaling phenotype. NH2-terminally truncated ß1ARs differ from full-length ß1ARs in their signaling bias to cAMP/PKA versus ERK pathways and only the NH2-terminally truncated form of the ß1AR constitutively activates AKT and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes. Since the R31 ↓ L32 sequence conforms to a trypsin consensus cleavage site, we used immunoblotting methods to test the hypothesis that ß1ARs are also cleaved at R31 ↓ L32 by trypsin (an enzyme typically used to isolate cardiomyocytes from the intact ventricle). We show that full-length ß1ARs are cleaved by trypsin and that trypsin cleaves the full-length ß1AR NH2-terminus specifically at R31 ↓ L32 in CHO-Pro5 cells. Trypsin also cleaves ß1ARs in cardiomyocytes, but at a second site that results in the formation of ∼40-kDa NH2-terminal and ∼30-kDa COOH-terminal fragments. The observation that cardiomyocyte ß1ARs are cleaved by trypsin (a mechanism that constitutes a heretofore-unrecognized mechanism that would influence ß1AR-signaling responses) suggests that studies that use standard trypsin-based procedures to isolate adult cardiomyocytes from the intact ventricle should be interpreted with caution.NEW & NOTEWORTHY Current concepts regarding the molecular basis for ß1AR responses derive from literature predicated on the assumption that ß1ARs signal exclusively as full-length receptor proteins. However, we recently showed that ß1ARs accumulate as both full-length and NH2-terminally truncated forms. This manuscript provides novel evidence that ß1-adrenergic receptors can be cleaved by trypsin and that cell surface ß1AR cleavage constitutes a heretofore unrecognized mechanism to alter catecholamine-dependent signaling responses.

N-Tertaining a New Signaling Paradigm for the Cardiomyocyte ß 1 -Adrenergic Receptor.

ABSTRACT: ß 1 -adrenergic receptors (ß 1 ARs) are the principle mediators of catecholamine actions in cardiomyocytes. ß 1 ARs rapidly adjust cardiac output and provide short-term hemodynamic support for the failing heart by activating a Gs-adenylyl cyclase pathway that increases 3'-5'-cyclic adenosine monophosphate and leads to the activation of protein kinase A and the phosphorylation of substrates involved in excitation-contraction coupling. However, chronic persistent ß 1 AR activation in the setting of heart failure leads to a spectrum of maladaptive changes that contribute to the evolution of heart failure. The molecular basis for ß 1 AR-driven maladaptive responses remains uncertain because chronic persistent ß 1 AR activation has been linked to the activation of both proapoptotic and antiapoptotic signaling pathways. Of note, studies to date have been predicated on the assumption that ß 1 ARs signal exclusively as full-length receptor proteins. Our recent studies show that ß 1 ARs are detected as both full-length and N-terminally truncated species in cardiomyocytes, that N-terminal cleavage is regulated by O-glycan modifications at specific sites on the ß 1 AR N-terminus, and that N-terminally truncated ß 1 ARs remain signaling competent, but their signaling properties differ from those of the full-length ß 1 AR. The N-terminally truncated form of the ß 1 AR constitutively activates the protein kinase B signaling pathway and confers protection against doxorubicin-dependent apoptosis in cardiomyocytes. These studies identify a novel signaling paradigm for the ß 1 AR, implicating the N-terminus as a heretofore-unrecognized structural determinant of ß 1 AR responsiveness that could be pharmacologically targeted for therapeutic advantage.

Decoding the Cardiac Actions of Protein Kinase D Isoforms.

Protein kinase D (PKD) consists of a family of three structurally related enzymes that play key roles in a wide range of biological functions that contribute to the evolution of cardiac hypertrophy and heart failure. PKD1 (the founding member of this enzyme family) has been implicated in the phosphorylation of substrates that regulate cardiac hypertrophy, contraction, and susceptibility to ischemia/reperfusion injury, and de novo PRKD1 (protein kinase D1 gene) mutations have been identified in patients with syndromic congenital heart disease. However, cardiomyocytes coexpress all three PKDs. Although stimulus-specific activation patterns for PKD1, PKD2, and PKD3 have been identified in cardiomyocytes, progress toward identifying PKD isoform-specific functions in the heart have been hampered by significant gaps in our understanding of the molecular mechanisms that regulate PKD activity. This review incorporates recent conceptual breakthroughs in our understanding of various alternative mechanisms for PKD activation, with an emphasis on recent evidence that PKDs activate certain effector responses as dimers, to consider the role of PKD isoforms in signaling pathways that drive cardiac hypertrophy and ischemia/reperfusion injury. The focus is on whether the recently identified activation mechanisms that enhance the signaling repertoire of PKD family enzymes provide novel therapeutic strategies to target PKD enzymes and prevent or slow the evolution of cardiac injury and pathological cardiac remodeling. SIGNIFICANCE STATEMENT: PKD isoforms regulate a large number of fundamental biological processes, but the understanding of the biological actions of individual PKDs (based upon studies using adenoviral overexpression or gene-silencing methods) remains incomplete. This review focuses on dimerization, a recently identified mechanism for PKD activation, and the notion that this mechanism provides a strategy to develop novel PKD-targeted pharmaceuticals that restrict proliferation, invasion, or angiogenesis in cancer and prevent or slow the evolution of cardiac injury and pathological cardiac remodeling.

A zebrafish forward genetic screen identifies an indispensable threonine residue in the kinase domain of PRKD2.

Protein kinase D2 belongs to a family of evolutionarily conserved enzymes regulating several biological processes. In a forward genetic screen for zebrafish cardiovascular mutants, we identified a mutation in the prkd2 gene. Homozygous mutant embryos develop as wild type up to 36 h post-fertilization and initiate blood flow, but fail to maintain it, resulting in a complete outflow tract stenosis. We identified a mutation in the prkd2 gene that results in a T757A substitution at a conserved residue in the kinase domain activation loop (T714A in human PRKD2) that disrupts catalytic activity and drives this phenotype. Homozygous mutants survive without circulation for several days, allowing us to study the extreme phenotype of no intracardiac flow, in the background of a functional heart. We show dysregulation of atrioventricular and outflow tract markers in the mutants and higher sensitivity to the Calcineurin inhibitor, Cyclosporin A. Finally we identify TBX5 as a potential regulator of PRKD2. Our results implicate PRKD2 catalytic activity in outflow tract development in zebrafish.This article has an associated First Person interview with the first author of the paper.

ß1-adrenergic receptor N-terminal cleavage by ADAM17; the mechanism for redox-dependent downregulation of cardiomyocyte ß1-adrenergic receptors.

ß1-adrenergic receptors (ß1ARs) are the principle mediators of catecholamine action in cardiomyocytes. We previously showed that the ß1AR extracellular N-terminus is a target for post-translational modifications that impact on signaling responses. Specifically, we showed that the ß1AR N-terminus carries O-glycan modifications at Ser37/Ser41, that O-glycosylation prevents ß1AR N-terminal cleavage, and that N-terminal truncation influences ß1AR signaling to downstream effectors. However, the site(s) and mechanism for ß1AR N-terminal cleavage in cells was not identified. This study shows that ß1ARs are expressed in cardiomyocytes and other cells types as both full-length and N-terminally truncated species and that the truncated ß1AR species is formed as a result of an O-glycan regulated N-terminal cleavage by ADAM17 at R31↓L32. We identify Ser41 as the major O-glycosylation site on the ß1AR N-terminus and show that an O-glycan modification at Ser41 prevents ADAM17-dependent cleavage of the ß1-AR N-terminus at S41↓L42, a second N-terminal cleavage site adjacent to this O-glycan modification (and it attenuates ß1-AR N-terminal cleavage at R31↓L32). We previously reported that oxidative stress leads to a decrease in ß1AR expression and catecholamine responsiveness in cardiomyocytes. This study shows that redox-inactivation of cardiomyocyte ß1ARs is via a mechanism involving N-terminal truncation at R31↓L32 by ADAM17. In keeping with the previous observation that N-terminally truncated ß1ARs constitutively activate an AKT pathway that affords protection against doxorubicin-dependent apoptosis, overexpression of a cleavage resistant ß1AR mutant exacerbates doxorubicin-dependent apoptosis. These studies identify the ß1AR N-terminus as a structural determinant of ß1AR responses that can be targeted for therapeutic advantage.

Telangiectasia-ectodermal dysplasia-brachydactyly-cardiac anomaly syndrome is caused by de novo mutations in protein kinase D1.

BACKGROUND: We describe two unrelated patients who display similar clinical features including telangiectasia, ectodermal dysplasia, brachydactyly and congenital heart disease. METHODS: We performed trio whole exome sequencing and functional analysis using in vitro kinase assays with recombinant proteins. RESULTS: We identified two different de novo mutations in protein kinase D1 (PRKD1, NM_002742.2): c.1774G>C, p.(Gly592Arg) and c.1808G>A, p.(Arg603His), one in each patient. PRKD1 (PKD1, HGNC:9407) encodes a kinase that is a member of the protein kinase D (PKD) family of serine/threonine protein kinases involved in diverse cellular processes such as cell differentiation and proliferation and cell migration as well as vesicle transport and angiogenesis. Functional analysis using in vitro kinase assays with recombinant proteins showed that the mutation c.1808G>A, p.(Arg603His) represents a gain-of-function mutation encoding an enzyme with a constitutive, lipid-independent catalytic activity. The mutation c.1774G>C, p.(Gly592Arg) in contrast shows a defect in substrate phosphorylation representing a loss-of-function mutation. CONCLUSION: The present cases represent a syndrome, which associates symptoms from several different organ systems: skin, teeth, bones and heart, caused by heterozygous de novo mutations in PRKD1 and expands the clinical spectrum of PRKD1 mutations, which have hitherto been linked to syndromic congenital heart disease and limb abnormalities.

Carvedilol Prevents Redox Inactivation of Cardiomyocyte Β1-Adrenergic Receptors.

The mechanism that leads to a decrease in ß1-adrenergic receptor (ß1AR) expression in the failing heart remains uncertain. This study shows that cardiomyocyte ß1AR expression and isoproterenol responsiveness decrease in response to oxidative stress. Studies of mechanisms show that the redox-dependent decrease in ß1AR expression is uniquely prevented by carvedilol and not other ßAR ligands. Carvedilol also promotes the accumulation of N-terminally truncated ß1ARs that confer protection against doxorubicin-induced apoptosis in association with activation of protein kinase B. The redox-induced molecular controls for cardiomyocyte ß1ARs and pharmacologic properties of carvedilol identified in this study have important clinical and therapeutic implications.

Post-translational modifications at the ATP-positioning G-loop that regulate protein kinase activity.

Protein kinases are a superfamily of enzymes that control a wide range of cellular functions. These enzymes share a highly conserved catalytic core that folds into a similar bilobar three-dimensional structure. One highly conserved region in the protein kinase core is the glycine-rich loop (or G-loop), a highly flexible loop that is characterized by a consensus GxGxxG sequence. The G-loop points toward the catalytic cleft and functions to bind and position ATP for phosphotransfer. Of note, in many protein kinases, the second and third glycine residues in the G-loop triad flank residues that can be targets for phosphorylation (Ser, Thr, or Tyr) or other post-translational modifications (ubiquitination, acetylation, O-GlcNAcylation, oxidation). There is considerable evidence that cyclin-dependent kinases are held inactive through inhibitory phosphorylation of the conserved Thr/Tyr residues in this position of the G-loop and that dephosphorylation by cellular phosphatases is required for CDK activation and progression through the cell cycle. This review summarizes literature that identifies residues in or adjacent to the G-loop in other protein kinases that are targets for functionally important post-translational modifications.

ß1-adrenergic receptor O-glycosylation regulates N-terminal cleavage and signaling responses in cardiomyocytes.

ß1-adrenergic receptors (ß1ARs) mediate catecholamine actions in cardiomyocytes by coupling to both Gs/cAMP-dependent and Gs-independent/growth-regulatory pathways. Structural studies of the ß1AR define ligand-binding sites in the transmembrane helices and effector docking sites at the intracellular surface of the ß1AR, but the extracellular N-terminus, which is a target for post-translational modifications, typically is ignored. This study identifies ß1AR N-terminal O-glycosylation at Ser37/Ser41 as a mechanism that prevents ß1AR N-terminal cleavage. We used an adenoviral overexpression strategy to show that both full-length/glycosylated ß1ARs and N-terminally truncated glycosylation-defective ß1ARs couple to cAMP and ERK-MAPK signaling pathways in cardiomyocytes. However, a glycosylation defect that results in N-terminal truncation stabilizes ß1ARs in a conformation that is biased toward the cAMP pathway. The identification of O-glycosylation and N-terminal cleavage as novel structural determinants of ß1AR responsiveness in cardiomyocytes could be exploited for therapeutic advantage.

Cleavage Alters the Molecular Determinants of Protein Kinase C-δ Catalytic Activity.

Protein kinase C-δ (PKCδ) is an allosterically activated enzyme that acts much like other PKC isoforms to transduce growth factor-dependent signaling responses. However, PKCδ is unique in that activation loop (Thr507) phosphorylation is not required for catalytic activity. Since PKCδ can be proteolytically cleaved by caspase-3 during apoptosis, the prevailing assumption has been that the kinase domain fragment (δKD) freed from autoinhibitory constraints imposed by the regulatory domain is catalytically competent and that Thr507 phosphorylation is not required for δKD activity. This study provides a counternarrative showing that δKD activity is regulated through Thr507 phosphorylation. We show that Thr507-phosphorylated δKD is catalytically active and not phosphorylated at Ser359 in its ATP-positioning G-loop. In contrast, a δKD fragment that is not phosphorylated at Thr507 (which accumulates in doxorubicin-treated cardiomyocytes) displays decreased C-terminal tail priming-site phosphorylation, increased G-loop Ser359 phosphorylation, and defective kinase activity. δKD is not a substrate for Src, but Src phosphorylates δKD-T507A at Tyr334 (in the newly exposed δKD N terminus), and this (or an S359A substitution) rescues δKD-T507A catalytic activity. These results expose a unique role for δKD-Thr507 phosphorylation (that does not apply to full-length PKCδ) in structurally organizing diverse elements within the enzyme that critically regulate catalytic activity.

Phos-tag SDS-PAGE resolves agonist- and isoform-specific activation patterns for PKD2 and PKD3 in cardiomyocytes and cardiac fibroblasts.

Protein kinase D (PKD) consists of a family of three structurally related enzymes that are co-expressed in the heart and have important roles in many biological responses. PKD1 is activated by pro-hypertrophic stimuli and has been implicated in adverse cardiac remodeling. Efforts to define the cardiac actions of PKD2 and PKD3 have been less successful at least in part because conventional methods provide a general screen for PKD activation but are poorly suited to resolve activation patterns for PKD2 or PKD3. This study uses Phos-tag SDS-PAGE, a method that exaggerates phosphorylation-dependent mobility shifts, to overcome this technical limitation. Phos-tag SDS-PAGE resolves PKD1 as distinct molecular species (indicative of pools of enzyme with distinct phosphorylation profiles) in unstimulated cardiac fibroblasts and cardiomyocytes; as a result, attempts to track PKD1 mobility shifts that result from agonist activation were only moderately successful. In contrast, PKD2 and PKD3 are recovered from resting cardiac fibroblasts and cardiomyocytes as single molecular species; both enzymes display robust mobility shifts in Phos-tag SDS-PAGE in response to treatment with sphingosine-1-phosphate, thrombin, PDGF, or H2O2. Studies with GF109203X implicate protein kinase C activity in the stimulus-dependent pathways that activate PKD2/PKD3 in both cardiac fibroblasts and cardiomyocytes. Studies with C3 toxin identify a novel role for Rho in the sphingosine-1-phosphate and thrombin receptor-dependent pathways that lead to the phosphorylation of PKD2/3 and the downstream substrate CREB in cardiomyocytes. In conclusion, Phos-tag SDS-PAGE provides a general screen for stimulus-specific changes in PKD2 and PKD3 phosphorylation and exposes a novel role for these enzymes in specific stress-dependent pathways that influence cardiac remodeling.

Protein kinase C mechanisms that contribute to cardiac remodelling.

Protein phosphorylation is a highly-regulated and reversible process that is precisely controlled by the actions of protein kinases and protein phosphatases. Factors that tip the balance of protein phosphorylation lead to changes in a wide range of cellular responses, including cell proliferation, differentiation and survival. The protein kinase C (PKC) family of serine/threonine kinases sits at nodal points in many signal transduction pathways; PKC enzymes have been the focus of considerable attention since they contribute to both normal physiological responses as well as maladaptive pathological responses that drive a wide range of clinical disorders. This review provides a background on the mechanisms that regulate individual PKC isoenzymes followed by a discussion of recent insights into their role in the pathogenesis of diseases such as cancer. We then provide an overview on the role of individual PKC isoenzymes in the regulation of cardiac contractility and pathophysiological growth responses, with a focus on the PKC-dependent mechanisms that regulate pump function and/or contribute to the pathogenesis of heart failure.

A novel phosphorylation site at Ser130 adjacent to the pseudosubstrate domain contributes to the activation of protein kinase C-δ.

Protein kinase C-δ (PKCδ) is a signalling kinase that regulates many cellular responses. Although most studies focus on allosteric mechanisms that activate PKCδ at membranes, PKCδ also is controlled via multi-site phosphorylation [Gong et al. (2015) Mol. Cell. Biol. 35: , 1727-1740]. The present study uses MS-based methods to identify PKCδ phosphorylation at Thr(50) and Ser(645) (in resting and PMA-treated cardiomyocytes) as well as Thr(37), Thr(38), Ser(130), Thr(164), Thr(211), Thr(215), Ser(218), Thr(295), Ser(299) and Thr(656) (as sites that increase with PMA). We focused on the consequences of phosphorylation at Ser(130) and Thr(141) (sites just N-terminal to the pseudosubstrate domain). We show that S130D and T141E substitutions co-operate to increase PKCδ's basal lipid-independent activity and that Ser(130)/Thr(141) di-phosphorylation influences PKCδ's substrate specificity. We recently reported that PKCδ preferentially phosphorylates substrates with a phosphoacceptor serine residue and that this is due to constitutive phosphorylation at Ser(357), an ATP-positioning G-loop site that limits PKCδ's threonine kinase activity [Gong et al. (2015) Mol. Cell. Biol. 35: , 1727-1740]. The present study shows that S130D and T141E substitutions increase PKCδ's threonine kinase activity indirectly by decreasing G loop phosphorylation at Ser(357). A S130F substitution [that mimics a S130F single-nt polymorphism (SNP) identified in some human populations] also increases PKCδ's maximal lipid-dependent catalytic activity and confers threonine kinase activity. Finally, we show that Ser(130)/Thr(141) phosphorylations relieve auto-inhibitory constraints that limit PKCδ's activity and substrate specificity in a cell-based context. Since phosphorylation sites map to similar positions relative to the pseudosubstrate domains of other PKCs, our results suggest that phosphorylation in this region of the enzyme may constitute a general mechanism to control PKC isoform activity.