Loss-of-function mutations in co-chaperone BAG3 destabilize small HSPs and cause cardiomyopathy.

Defective protein quality control (PQC) systems are implicated in multiple diseases. Molecular chaperones and co-chaperones play a central role in functioning PQC. Constant mechanical and metabolic stress in cardiomyocytes places great demand on the PQC system. Mutation and downregulation of the co-chaperone protein BCL-2-associated athanogene 3 (BAG3) are associated with cardiac myopathy and heart failure, and a BAG3 E455K mutation leads to dilated cardiomyopathy (DCM). However, the role of BAG3 in the heart and the mechanisms by which the E455K mutation leads to DCM remain obscure. Here, we found that cardiac-specific Bag3-KO and E455K-knockin mice developed DCM. Comparable phenotypes in the 2 mutants demonstrated that the E455K mutation resulted in loss of function. Further experiments revealed that the E455K mutation disrupted the interaction between BAG3 and HSP70. In both mutants, decreased levels of small heat shock proteins (sHSPs) were observed, and a subset of proteins required for cardiomyocyte function was enriched in the insoluble fraction. Together, these observations suggest that interaction between BAG3 and HSP70 is essential for BAG3 to stabilize sHSPs and maintain cardiomyocyte protein homeostasis. Our results provide insight into heart failure caused by defects in BAG3 pathways and suggest that increasing BAG3 protein levels may be of therapeutic benefit in heart failure.

Lmo7 is dispensable for skeletal muscle and cardiac function.

Emery-Dreifuss muscular dystrophy (EDMD) is a degenerative disease primarily affecting skeletal muscles in early childhood as well as cardiac muscle at later stages. EDMD is caused by a number of mutations in genes encoding proteins associated with the nuclear envelope (e.g., Emerin, Lamin A/C, and Nesprin). Recently, a novel protein, Lim-domain only 7 (lmo7) has been reported to play a role in the molecular pathogenesis of EDMD. Prior in vitro and in vivo studies suggested the intriguing possibility that Lmo7 plays a role in skeletal or cardiac muscle pathophysiology. To further understand the in vivo role of Lmo7 in striated muscles, we generated a novel Lmo7-null (lmo7(-/-)) mouse line. Using this mouse line, we examined skeletal and cardiac muscle physiology, as well as the role of Lmo7 in a model of muscular dystrophy and regeneration using the dystrophin-deficient mdx mouse model. Our results demonstrated that lmo7(-/-) mice had no abnormalities in skeletal muscle morphology, physiological function, or regeneration. Cardiac function was also unaffected. Moreover, we found that ablation of lmo7 in mdx mice had no effect on the observed myopathy and muscular regeneration exhibited by mdx mice. Molecular analyses also showed no changes in dystrophin complex factors, MAPK pathway components, and Emerin levels in lmo7 knockout mice. Taken together, we conclude that Lmo7 is dispensable for skeletal muscle and cardiac physiology and pathophysiology.

Novel Epac fluorescent ligand reveals distinct Epac1 vs. Epac2 distribution and function in cardiomyocytes.

Exchange proteins directly activated by cAMP (Epac1 and Epac2) have been recently recognized as key players in ß-adrenergic-dependent cardiac arrhythmias. Whereas Epac1 overexpression can lead to cardiac hypertrophy and Epac2 activation can be arrhythmogenic, it is unknown whether distinct subcellular distribution of Epac1 vs. Epac2 contributes to differential functional effects. Here, we characterized and used a novel fluorescent cAMP derivate Epac ligand 8-[Pharos-575]-2'-O-methyladenosine-3',5'-cyclic monophosphate (Φ-O-Me-cAMP) in mice lacking either one or both isoforms (Epac1-KO, Epac2-KO, or double knockout, DKO) to assess isoform localization and function. Fluorescence of Φ-O-Me-cAMP was enhanced by binding to Epac. Unlike several Epac-specific antibodies tested, Φ-O-Me-cAMP exhibited dramatically reduced signals in DKO myocytes. In WT, the apparent binding affinity (Kd = 10.2 ± 0.8 µM) is comparable to that of cAMP and nonfluorescent Epac-selective agonist 8-(4-chlorophenylthio)-2-O-methyladenosine-3'-,5'-cyclicmonophosphate (OMe-CPT). Φ-O-Me-cAMP readily entered intact myocytes, but did not activate PKA and its binding was competitively inhibited by OMe-CPT, confirming its Epac specificity. Φ-O-Me-cAMP is a weak partial agonist for purified Epac, but functioned as an antagonist for four Epac signaling pathways in myocytes. Epac2 and Epac1 were differentially concentrated along T tubules and around the nucleus, respectively. Epac1-KO abolished OMe-CPT-induced nuclear CaMKII activation and export of transcriptional regulator histone deacetylase 5. In conclusion, Epac1 is localized and functionally involved in nuclear signaling, whereas Epac2 is located at the T tubules and regulates arrhythmogenic sarcoplasmic reticulum Ca leak.

Development of a enterovirus diagnostic assay system for diagnosis of viral myocarditis in humans.

The coxsackieviruses type B3 (CVB3) are members of the genus Enterovirus of the family Picornaviridae. They are the commonest cause of chronic myocarditis and dilated cardiomyopathy. However, there is still no effective method for diagnosing CVB3 infection in humans. Here, a fast and accurate system that uses a capsid-protein-specific peptide sequence to detect CVB3 in the sera of patients with viral myocarditis was established. The peptide sequence was selected from the whole CVB3 capsid protein sequence by computationally predicting fragments with high antigenicity and low hydrophobicity. Two of eight possible peptide sequences were selected and commercially synthesized. The synthesized peptides encoded either the VP2 or VP1 capsid protein and induced immunoglobulin G antibody expression in immunized rabbits. Anti-VP2 and anti-VP1 sera detected the viral proteins extracted from CVB3-infected HeLa cells. The newly synthesized peptides successfully induced antibody production. These peptides, applied in an ELISA system, detected anti-CVB3 antibodies in virus-infected mouse serum. Moreover, an ELISA system based on the VP2 peptide detected CVB3 infection in patients with positively identified CVB3-induced fulminant myocarditis. These results indicate that these new peptides specifically interact with anti-CVB3 IgG antibodies in mouse and human sera. This ELISA system should be useful for the clinical diagnosis of enterovirus-induced myocarditis.

Epac2 mediates cardiac ß1-adrenergic-dependent sarcoplasmic reticulum Ca2+ leak and arrhythmia.

BACKGROUND: ß-Adrenergic receptor (ß-AR) activation can provoke cardiac arrhythmias mediated by cAMP-dependent alterations of Ca(2+) signaling. However, cAMP can activate both protein kinase A and an exchange protein directly activated by cAMP (Epac), but their functional interaction is unclear. In heart, selective Epac activation can induce potentially arrhythmogenic sarcoplasmic reticulum (SR) Ca(2+) release that involves Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) effects on the ryanodine receptor (RyR). METHODS AND RESULTS: We tested whether physiological ß-AR activation causes Epac-mediated SR Ca(2+) leak and arrhythmias and whether it requires Epac1 versus Epac2, ß(1)-AR versus ß(2)-AR, and CaMKIIδ-dependent phosphorylation of RyR2-S2814. We used knockout (KO) mice for Epac1, Epac2, or both. All KOs exhibited unaltered basal cardiac function, Ca(2+) handling, and hypertrophy in response to pressure overload. However, SR Ca(2+) leak induced by the specific Epac activator 8-CPT in wild-type mice was abolished in Epac2-KO and double-KO mice but was unaltered in Epac1-KO mice. ß-AR-induced arrhythmias were also less inducible in Epac2-KO versus wild-type mice. ß-AR activation with protein kinase A inhibition mimicked 8-CPT effects on SR Ca(2+) leak and was prevented by blockade of ß(1)-AR but not ß(2)-AR. CaMKII inhibition (KN93) and genetic ablation of either CaMKIIδ or CaMKII phosphorylation on RyR2-S2814 prevented 8-CPT-induced SR Ca(2+) leak. CONCLUSIONS: ß(1)-AR activates Epac2 to induce SR Ca(2+) leak via CaMKIIδ-dependent phosphorylation of RyR2-S2814. This pathway contributes to ß-AR-induced arrhythmias and reduced cardiac function.

Enhanced leptin sensitivity, reduced adiposity, and improved glucose homeostasis in mice lacking exchange protein directly activated by cyclic AMP isoform 1.

The prototypic second messenger cyclic AMP (cAMP) is essential for controlling cellular metabolism, including glucose and lipid homeostasis. In mammals, the majority of cAMP functions are mediated by cAMP-dependent protein kinase (PKA) and exchange proteins directly activated by cAMP (Epacs). To explore the physiological functions of Epac1, we generated Epac1 knockout mice. Here we report that Epac1 null mutants have reduced white adipose tissue and reduced plasma leptin levels but display heightened leptin sensitivity. Epac1-deficient mice are more resistant to high-fat diet-induced obesity, hyperleptinemia, and glucose intolerance. Furthermore, pharmacological inhibition of Epac by use of an Epac-specific inhibitor reduces plasma leptin levels in vivo and enhances leptin signaling in organotypic hypothalamic slices. Taken together, our results demonstrate that Epac1 plays an important role in regulating adiposity and energy balance.

Tesk1 interacts with Spry2 to abrogate its inhibition of ERK phosphorylation downstream of receptor tyrosine kinase signaling.

The Sprouty (Spry) proteins function as inhibitors of the Ras-ERK pathway downstream of various receptor tyrosine kinases. In this study, we have identified Tesk1 (testicular protein kinase 1) as a novel regulator of Spry2 function. Endogenous Tesk1 and Spry2 exist in a complex in cell lines and mouse tissues. Tesk1 coexpression relocalizes Spry2 to vesicles including endosomes, inhibiting its translocation to membrane ruffles upon growth factor stimulation. Independent of its kinase activity, Tesk1 binding leads to a loss of Spry2 function as an inhibitor of ERK phosphorylation and reverses inhibition of basic fibroblast growth factor (bFGF)- and nerve growth factor-induced neurite outgrowth in PC12 cells by Spry2. Furthermore, depletion of endogenous Tesk1 in PC12 cells leads to a reduction in neurite outgrowth induced by bFGF. Tesk1 nullifies the inhibitory effect of Spry2 by abrogating its interaction with the adaptor protein Grb2 and interfering with its serine dephosphorylation upon bFGF and FGF receptor 1 stimulation by impeding its binding to the catalytic subunit of protein phosphatase 2A. A construct of Tesk1 that binds to Spry2 but does not localize to the vesicles does not interfere with its function, highlighting the importance of subcellular localization of Tesk1 in this context. Conversely, Tesk1 does not affect interaction of Spry2 with the E3 ubiquitin ligase, c-Cbl, and consequently, does not affect its inhibition of Cbl-mediated ubiquitination of the epidermal growth factor receptor. By selectively modulating the downstream effects of Spry2, Tesk1 may thus serve as a molecular determinant of the signaling outcome.

Direct binding of PP2A to Sprouty2 and phosphorylation changes are a prerequisite for ERK inhibition downstream of fibroblast growth factor receptor stimulation.

In the context of fibroblast growth factor (FGF) signaling, Sprouty2 (Spry2) is the most profound inhibitor of the Ras/ERK pathway as compared with other Spry isoforms. An exclusive, necessary, but cryptic PXXPXR motif in the C terminus of Spry2 is revealed upon stimulation. The activation of Spry2 appears to be linked to sequences in the N-terminal half of the protein and correlated with a bandshifting seen on SDS-PAGE. The band-shifting is likely caused by changes in the phosphorylation status of key Ser and Thr residues following receptor stimulation. Dephosphorylation of at least two conserved Ser residues (Ser-112 and Ser-115) within a conserved Ser/Thr sequence is accomplished upon stimulation by a phosphatase that binds to Spry2 around residues 50-60. We show that human Spry2 co-immunoprecipitates with both the catalytic and the regulatory subunits of protein phosphatase 2A (PP2A-C and PP2A-A, respectively) in cells upon FGF receptor (FGFR) activation. PP2A-A binds directly to Spry2, but not to Spry2Delta50-60 (Delta50-60), and the activity of PP2A increases with both FGF treatment and FGFR1 overexpression. c-Cbl and PP2A-A compete for binding centered around Tyr-55 on Spry2. We show that there are at least two distinct pools of Spry2, one that binds PP2A and another that binds c-Cbl. c-Cbl binding likely targets Spry2 for ubiquitin-linked destruction, whereas the phosphatase binding and activity are necessary to dephosphorylate specific Ser/Thr residues. The resulting change in tertiary structure enables the Pro-rich motif to be revealed with subsequent binding of Grb2, a necessary step for Spry2 to act as a Ras/ERK pathway inhibitor in FGF signaling.

A Src homology 3-binding sequence on the C terminus of Sprouty2 is necessary for inhibition of the Ras/ERK pathway downstream of fibroblast growth factor receptor stimulation.

Because the Sprouty (Spry) proteins were shown to be inhibitors of the mainstream Ras/ERK pathway, there has been considerable interest in ascertaining their mechanism of action especially since a possible role as tumor suppressors for these inhibitory proteins has been suggested. We compared the ability of the mammalian Spry isoforms to inhibit the Ras/ERK pathway in the context of fibroblast growth factor receptor (FGFR) signaling. Spry2 is considerably more inhibitory than Spry1 or Spry4, and this correlates with the binding to Grb2 via a C-terminal proline-rich sequence that is found exclusively on Spry2. This PXXPXR motif binds directly to the N-terminal Src homology domain 3 of Grb2, and when added onto the C terminus of Spry4 the resultant chimera inhibits the Ras/ERK pathway. The ability to inhibit neurite outgrowth in PC-12 cells correlates with the propensity of Spry isoforms and engineered constructs to inhibit the phosphorylation of ERK1/2. The PXXPXR motif is cryptic in unstimulated cells, and it is postulated that Spry2 undergoes a conformational change following FGFR stimulation, enabling the subsequent interaction with Grb2. We present evidence that Spry2 can compete with the RasGEF (guanine nucleotide exchange factor) SOS1 for binding to Grb2, resulting in the inhibition of phosphorylation of ERK1/2.

The cysteine-rich sprouty translocation domain targets mitogen-activated protein kinase inhibitory proteins to phosphatidylinositol 4,5-bisphosphate in plasma membranes.

Sprouty (Spry) proteins have been revealed as inhibitors of the Ras/mitogen-activated protein kinase (MAPK) cascade, a pathway crucial for developmental processes initiated by activation of various receptor tyrosine kinases. In COS-1 and Swiss 3T3 cells, all Spry isoforms translocate to the plasma membrane, notably ruffles, following activation. Here we show that microinjection of active Rac induced the translocation of Spry isoforms, indicating that the target of the Spry translocation domain (SpryTD) is downstream of active Rac. Targeted disruption of actin polymerization revealed that the SpryTD target appeared upstream of cytoskeletal rearrangements. Accumulated evidence indicated that phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] is the likely SpryTD target. Human Spry2TD (hSpry2TD) binds to PtdIns(4,5)P(2) in vesicle-binding assays. hSpry2TD colocalizes with the pleckstrin homology domain of phospholipase Cdelta, which binds PtdIns(4,5)P(2). The plasma membrane localization of hSpry2TD was abolished in ionomycin-treated MDCK cells or when PtdIns(4,5)P(2) was specifically dephosphorylated by overexpression of an engineered, green fluorescent protein-tagged inositol 5-phosphatase. Similarly, Spred, a novel Ras/MAPK inhibitor recently found to contain the conserved cysteine-rich SpryTD, also translocated to peripheral membranes and bound to PtdIns(4,5)P(2). Alignment of the Spry and Spred proteins led us to identify a translocation-defective point mutant, hSpry2 D252. Targeting of hSpry2 to PtdIns(4,5)P(2) was shown to be essential for the down-regulation of Ras/MAPK signaling.

Sprouty2 inhibits the Ras/MAP kinase pathway by inhibiting the activation of Raf.

Several genetic studies in Drosophila have shown that the dSprouty (dSpry) protein inhibits the Ras/mitogen-activated protein (MAP) kinase pathway induced by various activated receptor tyrosine kinase receptors, most notably those of the epidermal growth factor receptor (EGFR) and fibroblast growth factor receptor (FGFR). Currently, the mode of action of dSpry is unknown, and the point of inhibition remains controversial. There are at least four mammalian Spry isoforms that have been shown to co-express preferentially with FGFRs as compared with EGFRs. In this study, we investigated the effects of the various mammalian Spry isoforms on the Ras/MAP kinase pathway in cells overexpressing constitutively active FGFR1. hSpry2 was significantly more potent than mSpry1 or mSpry4 in inhibiting the Ras/MAP kinase pathway. Additional experiments indicated that full-length hSpry2 was required for its full potency. hSpry2 had no inhibitory effect on either the JNK or the p38 pathway and displayed no inhibition of FRS2 phosphorylation, Akt activation, and Ras activation. Constitutively active mutants of Ras, Raf, and Mek were employed to locate the prospective point of inhibition of hSpry2 downstream of activated Ras. Results from this study indicated that hSpry2 exerted its inhibitory effect at the level of Raf, which was verified in a Raf activation assay in an FGF signaling context.