Inactivation of the mitochondrial heat shock protein zim17 leads to aggregation of matrix hsp70s followed by pleiotropic effects on morphology and protein biogenesis

The biogenesis of mitochondrial matrix proteins involves the translocase of the outer membrane, the presequence translocase of the inner membrane and the presequence translocase-associated motor. The mitochondrial heat shock protein 70 (mtHsp70) forms the central core of the motor. Recent studies led to the identification of Zim17, a mitochondrial zinc finger motif protein that interacts with mtHsp70. Different views have been reported on the localization of Zim17 in the mitochondrial inner membrane or matrix. Depletion of Zim17 impairs several critical mitochondrial processes, leading to inhibition of protein import, defects of Fe/S protein biogenesis and aggregation of Hsp70s in the matrix. Additionally, we found that inactivation of Zim17 altered the morphology of mitochondria. These pleiotropic effects raise the question of the specific function of Zim17 in mitochondria. Here, we report that Zim17 is a heat shock protein of the mitochondrial matrix that is loosely associated with the inner membrane. To address the function of Zim17 in organello, we generated a temperature-sensitive mutant allele of the ZIM17 gene in yeast. Upon a short-term shift of the yeast mutant cells to a non-permissive temperature, matrix Hsp70s aggregated while protein import, Fe/S protein activity and mitochondrial morphology were not, or only mildly, affected. Only after a long-term shift to non-permissive temperature, were strong defects in protein import, Fe/S protein activity and mitochondrial morphology observed. These findings suggest that the heat shock protein Zim17 plays a specific role in preventing protein aggregation in the mitochondrial matrix, and that aggregation of Hsp70s causes pleiotropic effects on protein biogenesis and mitochondrial morphology.     

The yeast dynamin-like protein, Mgm1p, functions on the mitochondrial outer membrane to mediate mitochondrial inheritance

The mdm17 mutation causes temperature-dependent defects in mitochondrial inheritance, mitochondrial morphology, and the maintenance of mitochondrial DNA in the yeast Saccharomyces cerevisiae. Defects in mitochondrial transmission to daughter buds and changes in mitochondrial morphology were apparent within 30 min after shifting cells to 37 degrees C, while loss of the mitochondrial genome occurred after 4-24 h at the elevated temperature. The mdm17 lesion mapped to MGM1, a gene encoding a dynamin-like GTPase previously implicated in mitochondrial genome maintenance, and the cloned MGM1 gene complements all of the mdm17 mutant phenotypes. Cells with an mgm1-null mutation displayed aberrant mitochondrial inheritance and morphology. A version of mgm1 mutated in a conserved residue in the putative GTP-binding site was unable to complement any of the mutant defects. It also caused aberrant mitochondrial distribution and morphology when expressed at high levels in cells that also contained a wild-type copy of the gene. Mgm1p was localized to the mitochondrial outer membrane and fractionated as a component of a high molecular weight complex. These results indicate that Mgm1p is a mitochondrial inheritance and morphology component that functions on the mitochondrial surface.     

The role of the cysteine residue in the chaperone and anti‐apoptotic functions of human Hsp27

The small heat shock protein Hsp27 is a molecular chaperone and an anti‐apoptotic protein. Human Hsp27 has one cysteine residue at position 137. We investigated the role of this cysteine residue in the chaperone and anti‐apoptotic functions of Hsp27 by mutating the cysteine residue to an alanine (Hsp27_C137A) and comparing it to wild‐type protein (Hsp27_WT). Both proteins were multi‐subunit oligomers, but subunits of Hsp27_WT were disulfide‐linked unlike those of Hsp27_C137A, which were monomeric. Hsp27_C137A was indistinguishable from Hsp27_WT with regard to its secondary structure, surface hydrophobicity, oligomeric size and chaperone function. S‐thiolation and reductive methylation of the cysteine residue had no apparent effect on the chaperone function of Hsp27_WT. The anti‐apoptotic function of Hsp27_C137A and Hsp27_WT was studied by overexpressing them in CHO cells. No difference in the caspase‐3 or ‐9 activity was observed in staurosporine‐treated cells. The rate of apoptosis between Hsp27_C137A and Hsp27_WT overexpressing cells was similar whether the cells were treated with staurosporine or etoposide. However, the mutant protein was less protective relative to the wild‐type protein in preventing caspase‐3 and caspase‐9 activation and apoptosis induced by 1 mM H₂O₂ in CHO and HeLa cells. These data demonstrate that in human Hsp27, disulfide formation by the lone cysteine does not affect its chaperone function and anti‐apoptotic function against chemical toxicants. However, oxidation of the lone cysteine in Hsp27 might at least partially affect the anti‐apoptotic function against oxidative stress. J. Cell. Biochem. 110: 408–419, 2010. © 2010 Wiley‐Liss, Inc.     

The Functional Interaction of Mitochondrial Hsp70s with the Escort Protein Zim17 Is Critical for Fe/S Biogenesis and Substrate Interaction at the Inner Membrane Preprotein Translocase

The yeast protein Zim17 belongs to a unique class of co-chaperones that maintain the solubility of Hsp70 proteins in mitochondria and plastids of eukaryotic cells. However, little is known about the functional cooperation between Zim17 and mitochondrial Hsp70 proteins in vivo. To analyze the effects of a loss of Zim17 function in the authentic environment, we introduced novel conditional mutations within the ZIM17 gene of the model organism Saccharomyces cerevisiae that allowed a recovery of temperature-sensitive but respiratory competent zim17 mutant cells. On fermentable growth medium, the mutant cells were prone to acquire respiratory deficits and showed a strong aggregation of the mitochondrial Hsp70 Ssq1 together with a concomitant defect in Fe/S protein biogenesis. In contrast, under respiring conditions, the mitochondrial Hsp70s Ssc1 and Ssq1 exhibited only a partial aggregation. We show that the induction of the zim17 mutant phenotype leads to strong import defects for Ssc1-dependent matrix-targeted precursor proteins that correlate with a significantly reduced binding of newly imported substrate proteins to Ssc1. We conclude that Zim17 is not only required for the maintenance of mtHsp70 solubility but also directly assists the functional interaction of mtHsp70 with substrate proteins in a J-type co-chaperone-dependent manner.     

A1 adenosine receptor–stimulated exocytosis in bladder umbrella cells requires phosphorylation of ADAM17 Ser-811 and EGF receptor transactivation

Despite the importance of ADAM17-dependent cleavage in normal biology and disease, the physiological cues that trigger its activity, the effector pathways that promote its function, and the mechanisms that control its activity, particularly the role of phosphorylation, remain unresolved. Using native bladder epithelium, in some cases transduced with adenoviruses encoding small interfering RNA, we observe that stimulation of apically localized A₁ adenosine receptors (A₁ARs) triggers a G_i-G_βγ-phospholipase C-protein kinase C (PKC) cascade that promotes ADAM17-dependent HB-EGF cleavage, EGFR transactivation, and apical exocytosis. We further show that the cytoplasmic tail of rat ADAM17 contains a conserved serine residue at position 811, which resides in a canonical PKC phosphorylation site, and is phosphorylated in response to A₁AR activation. Preventing this phosphorylation event by expression of a nonphosphorylatable ADAM17^S811A mutant or expression of a tail-minus construct inhibits A₁AR-stimulated, ADAM17-dependent HB-EGF cleavage. Furthermore, expression of ADAM17^S811A in bladder tissues impairs A₁AR-induced apical exocytosis. We conclude that adenosine-stimulated exocytosis requires PKC- and ADAM17-dependent EGFR transactivation and that the function of ADAM17 in this pathway depends on the phosphorylation state of Ser-811 in its cytoplasmic domain.     

Regulation of Hsp27 oligomerization, chaperone function, and protective activity against oxidative stress/tumor necrosis factor alpha by phosphorylation.

The small heat shock proteins (sHsps) from human (Hsp27) and mouse (Hsp25) form large oligomers which can act as molecular chaperones in vitro and protect cells from heat shock and oxidative stress when overexpressed. In addition, mammalian sHsps are rapidly phosphorylated by MAPKAP kinase 2/3 at two or three serine residues in response to various extracellular stresses. Here we analyze the effect of sHsp phosphorylation on its quaternary structure, chaperone function, and protection against oxidative stress. We show that in vitro phosphorylation of recombinant sHsp as well as molecular mimicry of Hsp27 phosphorylation lead to a significant decrease of the oligomeric size. We demonstrate that both phosphorylated sHsps and the triple mutant Hsp27-S15D,S78D,S82D show significantly decreased abilities to act as molecular chaperones suppressing thermal denaturation and facilitating refolding of citrate synthase in vitro. In parallel, Hsp27 and its mutants were analyzed for their ability to confer resistance against oxidative stress when overexpressed in L929 and 13.S.1.24 cells. While wild type Hsp27 confers resistance, the triple mutant S15D,S78D,S82D cannot protect against oxidative stress effectively. These data indicate that large oligomers of sHsps are necessary for chaperone action and resistance against oxidative stress whereas phosphorylation down-regulates these activities by dissociation of sHsp complexes to tetramers.     

CaMKII‐dependent ryanodine receptor phosphorylation mediates sepsis‐induced cardiomyocyte apoptosis

Sepsis is associated with cardiac dysfunction, which is at least in part due to cardiomyocyte apoptosis. However, the underlying mechanisms are far from being understood. Using the colon ascendens stent peritonitis mouse model of sepsis (CASP), we examined the subcellular mechanisms that mediate sepsis‐induced apoptosis. Wild‐type (WT) CASP mice hearts showed an increase in apoptosis respect to WT‐Sham. CASP transgenic mice expressing a CaMKII inhibitory peptide (AC3‐I) were protected against sepsis‐induced apoptosis. Dantrolene, used to reduce ryanodine receptor (RyR) diastolic sarcoplasmic reticulum (SR) Ca²⁺ release, prevented apoptosis in WT‐CASP. To examine whether CaMKII‐dependent RyR2 phosphorylation mediates diastolic Ca²⁺ release and apoptosis in sepsis, we evaluated apoptosis in mutant mice hearts that have the CaMKII phosphorylation site of RyR2 (Serine 2814) mutated to Alanine (S2814A). S2814A CASP mice did not show increased apoptosis. Consistent with RyR2 phosphorylation‐dependent enhancement in diastolic SR Ca²⁺ release leading to mitochondrial Ca²⁺ overload, mitochondrial Ca²⁺ retention capacity was reduced in mitochondria isolated from WT‐CASP compared to Sham and this reduction was absent in mitochondria from CASP S2814A or dantrolene‐treated mice. We conclude that in sepsis, CaMKII‐dependent RyR2 phosphorylation results in diastolic Ca²⁺ release from SR which leads to mitochondrial Ca²⁺ overload and apoptosis.     

Assessing Glucose Uptake through the Yeast Hexose Transporter 1 (Hxt1)

The transport of glucose across the plasma membrane is mediated by members of the glucose transporter family. In this study, we investigated glucose uptake through the yeast hexose transporter 1 (Hxt1) by measuring incorporation of 2-NBDG, a non-metabolizable, fluorescent glucose analog, into the yeast Saccharomyces cerevisiae. We find that 2-NBDG is not incorporated into the hxt null strain lacking all glucose transporter genes and that this defect is rescued by expression of wild type Hxt1, but not of Hxt1 with mutations at the putative glucose-binding residues, inferred from the alignment of yeast and human glucose transporter sequences. Similarly, the growth defect of the hxt null strain on glucose is fully complemented by expression of wild type Hxt1, but not of the mutant Hxt1 proteins. Thus, 2-NBDG, like glucose, is likely to be transported into the yeast cells through the glucose transport system. Hxt1 is internalized and targeted to the vacuole for degradation in response to glucose starvation. Among the mutant Hxt1 proteins, Hxt1^N370A and HXT1^W473A are resistant to such degradation. Hxt1^N370A, in particular, is able to neither uptake 2-NBDG nor restore the growth defect of the hxt null strain on glucose. These results demonstrate 2-NBDG as a fluorescent probe for glucose uptake in the yeast cells and identify N370 as a critical residue for the stability and function of Hxt1.     

Modular Control of Cross-oligomerization: ANALYSIS OF SUPERSTABILIZED Hsp90 HOMODIMERS IN VIVO*

Homo-oligomeric proteins fulfill numerous functions in all cells. The ability to co-express subunits of these proteins that preferentially self-assemble without cross-oligomerizing provides for controlled experiments to analyze the function of mutant homo-oligomers in vivo. Hsp90 is a dimeric chaperone involved in the maturation of many kinases and steroid hormone receptors. We observed that co-expression of different Hsp90 subunits in Saccharomyces cerevisiae caused unpredictable synthetic growth defects due to cross-dimerization. We engineered superstabilized Hsp90 dimers that resisted cross-dimerization with endogenous Hsp90 and alleviated the synthetic growth defect. Superstabilized Hsp90 dimers supported robust growth of S. cerevisiae, indicating that dissociation of Hsp90 dimers could be hindered without compromising essential function. We utilized superstabilized dimers to analyze the activity of ATPase mutant homodimers in a temperature-sensitive yeast background where elevated temperature inactivated all other Hsp90 species. We found that ATP binding and hydrolysis by Hsp90 are both required for the efficient maturation of glucocorticoid receptor and v-Src, confirming the critical role of ATP hydrolysis in the maturation of steroid hormone receptors and kinases in vivo.     

The Legs at odd angles (Loa) Mutation in Cytoplasmic Dynein Ameliorates Mitochondrial Function in SOD1G93A Mouse Model for Motor Neuron Disease

Amyotrophic lateral sclerosis (ALS) is a debilitating and fatal late-onset neurodegenerative disease. Familial cases of ALS (FALS) constitute ∼10% of all ALS cases, and mutant superoxide dismutase 1 (SOD1) is found in 15–20% of FALS. SOD1 mutations confer a toxic gain of unknown function to the protein that specifically targets the motor neurons in the cortex and the spinal cord. We have previously shown that the autosomal dominant Legs at odd angles (Loa) mutation in cytoplasmic dynein heavy chain (Dync1h1) delays disease onset and extends the life span of transgenic mice harboring human mutant SOD1^G93A. In this study we provide evidence that despite the lack of direct interactions between mutant SOD1 and either mutant or wild-type cytoplasmic dynein, the Loa mutation confers significant reductions in the amount of mutant SOD1 protein in the mitochondrial matrix. Moreover, we show that the Loa mutation ameliorates defects in mitochondrial respiration and membrane potential observed in SOD1^G93A motor neuron mitochondria. These data suggest that the Loa mutation reduces the vulnerability of mitochondria to the toxic effects of mutant SOD1, leading to improved mitochondrial function in SOD1^G93A motor neurons.     

Pancreatic and duodenal homeobox 1 (PDX1) phosphorylation at serine-269 is HIPK2-dependent and affects PDX1 subnuclear localization

Pancreatic and duodenal homeobox 1 (PDX1) regulates pancreatic development and mature β-cell function. We demonstrate by mass spectrometry that serine residue at position 269 in the C-terminal domain of PDX1 is phosphorylated in β-cells. Besides we show that the degree of phosphorylation, assessed with a phospho-Ser-269-specific antibody, is decreased by elevated glucose concentrations in both MIN6 β-cells and primary mouse pancreatic islets. Homeodomain interacting protein kinase 2 (HIPK2) phosphorylates PDX1 in vitro; phosphate incorporation substantially decreases in PDX1 S269A mutant. Silencing of HIPK2 led to a 51 ± 0.2% decrease in Ser-269 phosphorylation in MIN6 β-cells. Mutation of Ser-269 to phosphomimetic residue glutamic acid (S269E) or de-phosphomimetic residue alanine (S269A) exerted no effect on PDX1 half-life. Instead, PDX1 S269E mutant displayed abnormal changes in subnuclear localization in response to high glucose. Our results suggest that HIPK2-mediated phosphorylation of PDX1 at Ser-269 might be a regulatory mechanism connecting signals generated by changes in extracellular glucose concentration to downstream effectors via changes in subnuclear localization of PDX1, thereby influencing islet cell differentiation and function.     

Structure and function of the N‐terminal domain of the human mitochondrial calcium uniporter

The mitochondrial calcium uniporter (MCU) is responsible for mitochondrial calcium uptake and homeostasis. It is also a target for the regulation of cellular anti‐/pro‐apoptosis and necrosis by several oncogenes and tumour suppressors. Herein, we report the crystal structure of the MCU N‐terminal domain (NTD) at a resolution of 1.50 Å in a novel fold and the S92A MCU mutant at 2.75 Å resolution; the residue S92 is a predicted CaMKII phosphorylation site. The assembly of the mitochondrial calcium uniporter complex (uniplex) and the interaction with the MCU regulators such as the mitochondrial calcium uptake‐1 and mitochondrial calcium uptake‐2 proteins (MICU1 and MICU2) are not affected by the deletion of MCU NTD. However, the expression of the S92A mutant or a NTD deletion mutant failed to restore mitochondrial Ca²⁺ uptake in a stable MCU knockdown HeLa cell line and exerted dominant‐negative effects in the wild‐type MCU‐expressing cell line. These results suggest that the NTD of MCU is essential for the modulation of MCU function, although it does not affect the uniplex formation.     

Insulin Resistance Prevents AMPK-induced Tau Dephosphorylation through Akt-mediated Increase in AMPKSer-485 Phosphorylation

Metabolic syndrome (MetS) is a cluster of cardiovascular risk factors including obesity, diabetes, and dyslipidemia, and insulin resistance (IR) is the central feature of MetS. Recent studies suggest that MetS is a risk factor for Alzheimer disease (AD). AMP-activated kinase (AMPK) is an evolutionarily conserved fuel-sensing enzyme and a key player in regulating energy metabolism. In this report, we examined the role of IR on the regulation of AMPK phosphorylation and AMPK-mediated Tau phosphorylation. We found that AMPK^Ser-485, but not AMPK^Thr-172, phosphorylation is increased in the cortex of db/db and high fat diet-fed obese mice, two mouse models of IR. In vitro, treatment of human cortical stem cell line (HK-5320) and primary mouse embryonic cortical neurons with the AMPK activator, 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR), induced AMPK phosphorylation at both Thr-172 and Ser-485. AMPK activation also triggered Tau dephosphorylation. When IR was mimicked in vitro by chronically treating the cells with insulin, AICAR specifically induced AMPK^Ser-485, but not AMPK^Thr-172, hyperphosphorylation whereas AICAR-induced Tau dephosphorylation was inhibited. IR also resulted in the overactivation of Akt by AICAR treatment; however, preventing Akt overactivation during IR prevented AMPK^Ser-485 hyperphosphorylation and restored AMPK-mediated Tau dephosphorylation. Transfection of AMPK^S485A mutant caused similar results. Therefore, our results suggest the following mechanism for the adverse effect of IR on AD pathology: IR → chronic overactivation of Akt → AMPK^Ser-485 hyperphosphorylation → inhibition of AMPK-mediated Tau dephosphorylation. Together, our results show for the first time a possible contribution of IR-induced AMPK^Ser-485 phosphorylation to the increased risk of AD in obesity and diabetes.     

PKD1 Inhibits AMPKα2 through Phosphorylation of Serine 491 and Impairs Insulin Signaling in Skeletal Muscle Cells

AMP-activated protein kinase (AMPK) is an energy-sensing enzyme whose activity is inhibited in settings of insulin resistance. Exposure to a high glucose concentration has recently been shown to increase phosphorylation of AMPK at Ser^485/491 of its α1/α2 subunit; however, the mechanism by which it does so is not known. Diacylglycerol (DAG), which is also increased in muscle exposed to high glucose, activates a number of signaling molecules including protein kinase (PK)C and PKD1. We sought to determine whether PKC or PKD1 is involved in inhibition of AMPK by causing Ser^485/491 phosphorylation in skeletal muscle cells. C2C12 myotubes were treated with the PKC/D1 activator phorbol 12-myristate 13-acetate (PMA), which acts as a DAG mimetic. This caused dose- and time-dependent increases in AMPK Ser^485/491 phosphorylation, which was associated with a ∼60% decrease in AMPKα2 activity. Expression of a phosphodefective AMPKα2 mutant (S491A) prevented the PMA-induced reduction in AMPK activity. Serine phosphorylation and inhibition of AMPK activity were partially prevented by the broad PKC inhibitor Gö6983 and fully prevented by the specific PKD1 inhibitor CRT0066101. Genetic knockdown of PKD1 also prevented Ser^485/491 phosphorylation of AMPK. Inhibition of previously identified kinases that phosphorylate AMPK at this site (Akt, S6K, and ERK) did not prevent these events. PMA treatment also caused impairments in insulin-signaling through Akt, which were prevented by PKD1 inhibition. Finally, recombinant PKD1 phosphorylated AMPKα2 at Ser⁴⁹¹ in cell-free conditions. These results identify PKD1 as a novel upstream kinase of AMPKα2 Ser⁴⁹¹ that plays a negative role in insulin signaling in muscle cells.     

The role of serine 190 in FOXO nuclear export and cell death induction in Drosophila melanogaster

Proteins in the forkhead box O (FOXO) family contain three Akt phosphorylation sites that are important for export of the protein from the nucleus to the cytosol. In mammalian FOXO1, phosphorylation of serine 256 (S256) is a prerequisite for the phosphorylation of the other two sites. Although Drosophila FOXO (dFOXO) contains three well-conserved Akt phosphorylation sites, their role in the regulation of Drosophila physiology is not well understood. In the present study, we examine the regulation and function of phosphorylation at serine 190 (S190), which corresponds to S256 of mammalian FOXO1. Insulin and Akt were shown to increase S190 phosphorylation of dFOXO. Moreover, dFOXO nuclear export was induced by insulin treatment in both fly tissues and transfected Drosophila and human cells, and a protein containing an alanine substitution at S190 (dFOXO^S190A) was defective in these insulin-dependent responses, suggesting that S190 phosphorylation is required for dFOXO nuclear export. Interestingly, dFOXO^S190A and dFOXO^S190D mutants showed lower target gene expression and a reduced ability to induce cell death compared to wild-type dFOXO. These results suggest that the S190 residue is required for dFOXO translocation and is important for the pro-apoptotic function of dFOXO.     

Hsp90 Nuclear Accumulation in Quiescence Is Linked to Chaperone Function and Spore Development in Yeast

The 90-kDa heat-shock protein (Hsp90) operates in the context of a multichaperone complex to promote maturation of nuclear and cytoplasmic clients. We have discovered that Hsp90 and the cochaperone Sba1/p23 accumulate in the nucleus of quiescent Saccharomyces cerevisiae cells. Hsp90 nuclear accumulation was unaffected in sba1Δ cells, demonstrating that Hsp82 translocates independently of Sba1. Translocation of both chaperones was dependent on the α/β importin SRP1/KAP95. Hsp90 nuclear retention was coincident with glucose exhaustion and seems to be a starvation-specific response, as heat shock or 10% ethanol stress failed to elicit translocation. We generated nuclear accumulation-defective HSP82 mutants to probe the nature of this targeting event and identified a mutant with a single amino acid substitution (I578F) sufficient to retain Hsp90 in the cytoplasm in quiescent cells. Diploid hsp82-I578F cells exhibited pronounced defects in spore wall construction and maturation, resulting in catastrophic sporulation. The mislocalization and sporulation phenotypes were shared by another previously identified HSP82 mutant allele. Pharmacological inhibition of Hsp90 with macbecin in sporulating diploid cells also blocked spore formation, underscoring the importance of this chaperone in this developmental program.     

High glucose induces Drp1-mediated mitochondrial fission via the Orai1 calcium channel to participate in diabetic cardiomyocyte hypertrophy

Mitochondrial dysfunction and impaired Ca²⁺ handling are involved in the development of diabetic cardiomyopathy (DCM). Dynamic relative protein 1 (Drp1) regulates mitochondrial fission by changing its level of phosphorylation, and the Orai1 (Ca²⁺ release-activated calcium channel protein 1) calcium channel is important for the increase in Ca²⁺ entry into cardiomyocytes. We aimed to explore the mechanism of Drp1 and Orai1 in cardiomyocyte hypertrophy caused by high glucose (HG). We found that Zucker diabetic fat rats induced by administration of a high-fat diet develop cardiac hypertrophy and impaired cardiac function, accompanied by the activation of mitochondrial dynamics and calcium handling pathway-related proteins. Moreover, HG induces cardiomyocyte hypertrophy, accompanied by abnormal mitochondrial morphology and function, and increased Orai1-mediated Ca²⁺ influx. Mechanistically, the Drp1 inhibitor mitochondrial division inhibitor 1 (Mdivi-1) prevents cardiomyocyte hypertrophy induced by HG by reducing phosphorylation of Drp1 at serine 616 (S616) and increasing phosphorylation at S637. Inhibition of Orai1 with single guide RNA (sgOrai1) or an inhibitor (BTP2) not only suppressed Drp1 activity and calmodulin-binding catalytic subunit A (CnA) and phosphorylated-extracellular signal-regulated kinase (p-ERK1/2) expression but also alleviated mitochondrial dysfunction and cardiomyocyte hypertrophy caused by HG. In addition, the CnA inhibitor cyclosporin A and p-ERK1/2 inhibitor U0126 improved HG-induced cardiomyocyte hypertrophy by promoting and inhibiting phosphorylation of Drp1 at S637 and S616, respectively. In summary, we identified Drp1 as a downstream target of Orai1-mediated Ca²⁺ entry, via activation by p-ERK1/2-mediated phosphorylation at S616 or CnA-mediated dephosphorylation at S637 in DCM. Thus, the Orai1–Drp1 axis is a novel target for treating DCM.Subject terms: Molecular biology, Cardiac hypertrophy, Pathogenesis     

Phosphorylation of the pheromone-responsive Gβ protein of Saccharomyces cerevisiae does not affect its mating-specific signaling function

The pheromone-responsive G_β subunit of Saccharomyces cerevisiae (encoded by STE4) is rapidly phosphorylated at multiple sites when yeast cells are exposed to mating pheromone. It has been shown that a mutant form of Ste4 lacking residues 310–346, ste4Δ310–346, cannot be phosphorylated, and that its expression leads to defects in recovery from pheromone stimulation. Based on these observations, it was proposed that phosphorylation of Ste4 is associated with an adaptive response to mating pheromone. In this study we used site-directed mutagenesis to create two phosphorylation null (Pho^?) alleles of STE4: ste4-T320?A/S335A and ste4-T322 A/S335A and ste4-T322A/S335A. When expressed in yeast, these mutant forms of Ste4 remained unphosphorylated upon pheromone stimulation. The elimination of Ste4 phosphorylation has no discernible effect on either signaling or adaptation. In addition, disruption of the FUS3 gene, which encodes a pheromone-specific MAP kinase, leads to partial loss of pheromone-induced Ste4 phosphorylation. Two-hybrid analysis suggests that the ste4Δ310–346 deletion mutant is impaired in its interaction with Gpa1, the pheromone-responsive G_α of yeast, whereas the Ste4-T320A/S335A mutant has normal affinity for Gpa1. Taken together, these results indicate that pheromone-induced phosphorylation of Ste4 is not an adaptive mechanism, and that the adaptive defect exhibited by the 310–346 deletion mutant is likely to be due to disruption of the interaction between Ste4 and Gpa1.     

Mutation of the Ser18 phosphorylation site on the sole <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> UCS protein,She4, can compromise high-temperature survival

Folding of the myosin head often requires the joint actions of Hsp90 and a dedicated UNC45, Cro1, She4 (UCS) domain-containing cochaperone protein. Relatively weak sequence conservation exists between the single UCS protein of simple eukaryotes (She4 in budding yeast) and the two UCS proteins of higher organisms (the general cell and smooth muscle UNC45s; UNC45-GC and UNC45-SM respectively). In vertebrates, UNC45-GC facilitates cytoskeletal function whereas the 55% identical UNC45-SM assists in the assembly of the contractile apparatus of cardiac and skeletal muscles. UNC45-SM, unlike UNC45-GC, shares with yeast She4 an IDSL sequence motif known to be a site of in vivo serine phosphorylation in yeast. Investigating this further, we found that both a non-phosphorylatable (S18A) and a phosphomimetic (S18E) mutant form of She4 could rescue the type 1 myosin localisation and endocytosis defects of the yeast she4Δ mutant at 39 °C. Nevertheless, at higher temperature (45 °C), only She4 (S18A), not She4(S18E), could substantially rescue the cell lysis defect of she4Δ mutant cells. In the yeast two-hybrid system, the non-phosphorylatable S18A and S251A mutant forms of She4 and UNC45-SM still displayed the stress-enhanced in vivo interaction with Hsp90 seen with the wild-type She4 and UNC45-SM. Such high-temperature enforcement to interaction was though lost with the phosphomimetic mutant forms (She4(S18E) and UNC45-SM (S251E)), an indication that phosphorylation might suppress these increases in She4/Hsp90 and UNC45-SM/Hsp90 interaction with stress.