Cyclic adenosine monophosphate-dependent phosphorylation of mammalian mitochondrial proteins: enzyme and substrate characterization and functional role.

A study is presented on cyclic adenosine monophosphate- (cAMP-) dependent phosphorylation of mammalian mitochondrial proteins. Immunodetection with specific antibodies reveals the presence of the catalytic and the regulatory subunits of cAMP-dependent protein kinase (PKA) in the inner membrane and matrix of bovine heart mitochondria. The mitochondrial cAMP-dependent protein kinase phosphorylates mitochondrial proteins of 29, 18, and 6.5 kDa. With added histone as substrate, PKA exhibits affinities for ATP and cAMP and pH optimum comparable to those of the cytosolic PKA. Among the mitochondrial proteins phosphorylated by PKA, one is the nuclear-encoded (NDUFS4 gene) 18 kDa subunit of complex I, which has phosphorylation consensus sites in the C terminus and in the presequence. cAMP promotes phosphorylation of the 18 kDa subunit of complex I in myoblasts in culture and in their isolated mitoplast fraction. In both cases cAMP-dependent phosphorylation of the 18 kDa subunit of complex I is accompanied by enhancement of the activity of the complex. These results, and the finding of mutations in the NDUFS4 gene in patients with complex I deficiency, provide evidence showing that cAMP-dependent phosphorylation of the 18 kDa subunit of complex I plays a major role in the control of the mitochondrial respiratory activity.     

The NDUFS4 nuclear gene of complex I of mitochondria and the cAMP cascade

Results of studies on the role of the 18 kDa (IP) polypeptide subunit of complex I, encoded by the nuclear NDUFS4 gene, in isolated bovine heart mitochondria and human and murine cell cultures are presented.The mammalian 18 kDa subunit has in the carboxy-terminal sequence a conserved consensus site (RVS), which in isolated mitochondria is phosphorylated by cAMP-dependent protein kinase (PKA). The catalytic and regulatory subunits of PKA have been directly immunodetected in the inner membrane/matrix fraction of mammalian mitochondria. In the mitochondrial inner membrane a PP2Cgamma-type phosphatase has also been immunodetected, which dephosphorylates the 18 kDa subunit, phosphorylated by PKA. This phosphatase is Mg(2+)-dependent and inhibited by Ca(2+). In human and murine fibroblast and myoblast cultures "in vivo", elevation of intracellular cAMP level promotes phosphorylation of the 18 kDa subunit and stimulates the activity of complex I and NAD-linked mitochondrial respiration.Four families have been found with different mutations in the cDNA of the NDUFS4 gene. These mutations, transmitted by autosomal recessive inheritance, were associated in homozygous children with fatal neurological syndrome. All these mutations destroyed the phosphorylation consensus site in the C terminus of the 18 kDa subunit, abolished cAMP activation of complex I and impaired its normal assembly.     

The human complex I NDUFS4 subunit: from gene structure to function and pathology

Complex I is the first and largest enzyme of the oxidative phosphorylation system. It consists of at least 43 subunits. Recent studies have shown that the NDUFS4 subunit of complex I contributes to the activation of the complex through cAMP dependent phosphorylation of a conserved site (RVS) located at the C-terminal region of this protein. This report focuses on the NDUFS4 subunit. Summarized is the current knowledge of this subunit, from gene structure to function and pathology.     

cAMP-dependent phosphorylation of the nuclear encoded 18-kDa (IP) subunit of respiratory complex I and activation of the complex in serum-starved mouse fibroblast cultures

A study is presented on the in vivo effect of elevated cAMP levels induced by cholera toxin on the phosphorylation of subunits of the mitochondrial respiratory complexes and their activities in Balb/c 3T3 mouse fibroblast cultures. Treatment of serum-starved fibroblasts with cholera toxin promoted serine phosphorylation in the 18-kDa subunit of complex I. Phosphorylation of the 18-kDa subunit, in response to cholera toxin treatment of fibroblasts, was accompanied by a 2-3-fold enhancement of the rotenone-sensitive endogenous respiration of fibroblasts, of the rotenone-sensitive NADH oxidase, and of the NADH:ubiquinone oxidoreductase activity of complex I. Direct exposure of fibroblasts to dibutyryl cAMP resulted in an equally potent stimulation of the NADH:ubiquinone oxidoreductase activity. Stimulation of complex I activity and respiration with NAD-linked substrates were also observed upon short incubation of isolated fibroblast mitoplasts with dibutyryl cAMP and ATP, which also promoted phosphorylation of the 18-kDa subunit. These observations document an extension of cAMP-mediated intracellular signal transduction to the regulation of cellular respiration.     

Contribution of the Phosphorylable Complex I in the Growth Phase-Dependent Respiration of C6 Glioma Cells in Vitro

The energy metabolism of rat C6 glioma cells was investigated as a function of the growth phases. Three-dimensional cultures of C6 cells exhibited diminished respiration and respiratory capacity during the early growth phase, before reaching confluence. This decrease in respiration was neither due to changes in the respiratory complex content nor in the mitochondrial mass per se. Nevertheless, a quantitative correlation was found between cellular respiration and the rotenone-sensitive NADH ubiquinone oxidoreductase (i.e. complex I) activity. Immunoblot analysis showed that phosphorylation of the 18 kDa-subunit of this complex was associated with the growth-phase dependent modulation of complex I and respiratory activity in C6 cells. In addition, by using forskolin or dibutyryl cAMP, short-term activation of protein kinases A of C6 cells correlated with increased phosphorylation of the 18-kDa subunit of complex I, activated NADH ubiquinone oxidoreductase activity and stimulated cellular respiration. These findings suggest that complex I of C6 glioma cells is a key regulating step that modulates the oxidative phosphorylation capacity during growth phase transitions.     

Activation of the cAMP cascade in human fibroblast cultures rescues the activity of oxidatively damaged complex I

A study of the relationship between cAMP/PKA-dependent phosphorylation and oxidative damage of subunits of complex I of the mitochondrial respiratory chain is presented. It is shown that, in fibroblast cultures, PKA-mediated phosphorylation of the NDUFS4 subunit of complex I rescues the activity of the oxidatively damaged complex. Evidence is presented showing that this effect is mediated by phosphorylation-dependent exchange of carbonylated NDUFS4 subunit in the assembled complex with the de novo synthesized subunit. These results indicate a potential use for β-adrenoceptor agonists in preventing/reversing the detrimental effects of oxidative stress in the mitochondrial respiratory system.     

The function of Mg-ATP in interactions between the regulatory and catalytic subunits of type I cAMP-dependent protein kinase from rabbit skeletal muscle

The regulatory subunit of Type I cAMP-dependent protein kinase from rabbit skeletal muscle can bind [3H]cAMP to form the R-[3H]cAMP complex, and the slow phase of the enhanced exchange of free cAMP with [3H]cAMP from the R-[3H]cAMP complexes was studied under various conditions using the equilibrium isotope exchange technique. Results indicate that Mg-ATP and the catalytic subunit are absolutely required for the enhanced exchange reaction to occur, but phosphorylation of the regulatory subunit by Mg-ATP does not play a determining role in the slow rate of the dissociation/association of the Type I protein-kinase in the presence of cAMP and the catalytic subunit. We interpret the role of Mg-ATP as being one in which it may provide the structural attributes required for formation of a stabilized transient state of the cAMP-regulatory subunit-catalytic subunit ternary complex, an obligatory intermediate involved in the dissociation/association of Type I cAMP-dependent protein kinase.     

A mammalian two pore domain mechano-gated S-like K+ channel.

Aplysia S-type K+ channels of sensory neurons play a dominant role in presynaptic facilitation and behavioural sensitization. They are closed by serotonin via cAMP-dependent phosphorylation, whereas they are opened by arachidonic acid, volatile general anaesthetics and mechanical stimulation. We have identified a cloned mammalian two P domain K+ channel sharing the properties of the S channel. In addition, the recombinant channel is opened by lipid bilayer amphipathic crenators, while it is closed by cup-formers. The cytoplasmic C-terminus contains a charged region critical for chemical and mechanical activation, as well as a phosphorylation site required for cAMP inhibition.     

昆虫鞣化激素研究进展

鞣化激素是调控昆虫体壁黑化及翅伸展的一类激素, 是由BURS和PBURS两个亚基组成的一种异源二聚体蛋白质。BURS和PBURS亚基在结构及其进化上相对较为保守, 氨基酸序列中均含有11个半胱氨酸残基。鞣化激素主要是在胸腹神经节中合成的, 一旦释放到血淋巴就与其受体LGR2结合进而激活cAMP/PKA信号, 从而促进酪氨酸羟化酶(tyrosine hydroxylase, TH)的磷酸化。活化后的TH将酪氨酸(tyrosine)转变为多巴（DOPA）, 引起昆虫表皮鞣化。同时, cAMP/PKA信号也引起翅真皮细胞凋亡从而促进翅的伸展。除了鞣化激素异聚体调控表皮鞣化及翅的伸展外, BURS亚基或PBURS亚基组成的同源二聚体经IMD路径, 激活转录因子Relish调控昆虫的免疫反应。本文就鞣化激素分子结构特性、 作用机制及功能等方面的研究进展进行了综述, 旨在为进一步研究昆虫鞣化激素提供借鉴和参考。     

Hh-induced Smoothened conformational switch is mediated by differential phosphorylation at its C-terminal tail in a dose- and position-dependent manner

The activation of Smoothened (Smo) requires phosphorylation at three clusters of Serine residues in Drosophila Hedgehog (Hh) signaling. However, the mechanism by which phosphorylation promotes Smo conformational change and subsequently activates Smo in response to Hh gradient remains unclear. Here, we show that the conformational states of Smo are determined by not only the amount but also the position of the negative charges provided by phosphorylation. By using a Smo phospho-specific antibody, we demonstrate that Smo is differentially phosphorylated at three clusters of serine residues in response to levels of Hh activity. Mutating the first cluster, compared to mutating the other clusters, impairs Smo activity more severely, whereas mutating the last cluster prohibits C-terminus dimerization. In addition, phosphorylation of the membrane proximal cluster promotes phosphorylation of the distal cluster. We propose a zipper-lock model in which the gradual phosphorylation at these clusters induces a gradual conformational change in the Smo cytoplasmic tail, which promotes the interaction between Smo and Costal2 (Cos2). Moreover, we show that Hh regulates both PKA and CK1 phosphorylation of Smo. Thus, the differential phosphorylation of Smo mediates the thresholds of Hh activity.     

Deficit of complex I activity in human skin fibroblasts with chromosome 21 trisomy and overproduction of reactive oxygen species by mitochondria: involvement of the cAMP/PKA signalling pathway

DS (Down's syndrome) is the most common human aneuploidy associated with mental retardation and early neurodegeneration. Mitochondrial dysfunction has emerged as a crucial factor in the pathogenesis of numerous neurological disorders including DS, but the cause of mitochondrial damage remains elusive. In the present study, we identified new molecular events involved in mitochondrial dysfunction which could play a role in DS pathogenesis. We analysed mitochondrial respiratory chain function in DS-HSFs (Down's syndrome human foetal skin fibroblasts; human foetal skin fibroblasts with chromosome 21 trisomy) and found a selective deficit in the catalytic efficiency of mitochondrial complex I. The complex I deficit was associated with a decrease in cAMP-dependent phosphorylation of the 18 kDa subunit of the complex, due to a decrease in PKA (protein kinase A) activity related to reduced basal levels of cAMP. Consistently, exposure of DS-HSFs to db-cAMP (dibutyryl-cAMP), a membrane-permeable cAMP analogue, stimulated PKA activity and consequently rescued the deficit of both the cAMP-dependent phosphorylation and the catalytic activity of complex I; conversely H89, a specific PKA inhibitor, suppressed these cAMP-dependent activations. Furthermore, in the present paper we report a 3-fold increase in cellular levels of ROS (reactive oxygen species), in particular superoxide anion, mainly produced by DS-HSF mitochondria. ROS accumulation was prevented by db-cAMP-dependent activation of complex I, suggesting its involvement in ROS production. Taken together, the results of the present study suggest that the drastic decrease in basal cAMP levels observed in DS-HSFs participates in the complex I deficit and overproduction of ROS by DS-HSF mitochondria.     

Functionally distinct serine phosphorylation sites of p36, the cellular substrate of retroviral protein kinase; differential inhibition of reassociation with p11.

P36 was originally defined as the major cytoplasmic target of retrovirally coded tyrosine-kinases. While recently much has been learned about its biochemistry, the functional importance of its tyrosine and serine phosphorylation has not been approached. As p36 is now understood as a multi-ligand protein its in vitro phosphorylation by three different serine/threonine kinases was followed. Monomeric p36 is a much better substrate than the complex containing two copies each of p36 and p11 (protein I). All p36 phosphorylation sites occur within the amino-terminal 29 residues specifically released by mild proteolysis. As this region harbors an important interaction site for p11 the reduced phosphorylation of p36 in the protein I complex results most likely from a lowered accessibility. Phosphorylation of p36 is serine specific. Reconstitution experiments define at least two functionally distinct sites. One product of protein kinase C reconstitutes with p11 to protein I, while this complex formation normal for p36 is observed neither for the second phosphorylation product nor for the derivatives resulting from phosphorylation by calmodulin or cAMP dependent kinases. The results lend direct support to the hypothesis that phosphorylation of p36 can modulate one of its molecular functions. Obvious implications for other Ca2+-dependent lipid binding proteins are discussed.     

Epigallocatechin-3-gallate prevents oxidative phosphorylation deficit and promotes mitochondrial biogenesis in human cells from subjects with Down's syndrome

A critical role for mitochondrial dysfunction has been proposed in the pathogenesis of Down's syndrome (DS), a human multifactorial disorder caused by trisomy of chromosome 21, associated with mental retardation and early neurodegeneration. Previous studies from our group demonstrated in DS cells a decreased capacity of the mitochondrial ATP production system and overproduction of reactive oxygen species (ROS) in mitochondria. In this study we have tested the potential of epigallocatechin-3-gallate (EGCG) – a natural polyphenol component of green tea – to counteract the mitochondrial energy deficit found in DS cells. We found that EGCG, incubated with cultured lymphoblasts and fibroblasts from DS subjects, rescued mitochondrial complex I and ATP synthase catalytic activities, restored oxidative phosphorylation efficiency and counteracted oxidative stress. These effects were associated with EGCG-induced promotion of PKA activity, related to increased cellular levels of cAMP and PKA-dependent phosphorylation of the NDUFS4 subunit of complex I. In addition, EGCG strongly promoted mitochondrial biogenesis in DS cells, as associated with increase in Sirt1-dependent PGC-1α deacetylation, NRF-1 and T-FAM protein levels and mitochondrial DNA content.In conclusion, this study shows that EGCG is a promoting effector of oxidative phosphorylation and mitochondrial biogenesis in DS cells, acting through modulation of the cAMP/PKA- and sirtuin-dependent pathways. EGCG treatment promises thus to be a therapeutic approach to counteract mitochondrial energy deficit and oxidative stress in DS.     

Regulation of Constitutive GPR3 Signaling and Surface Localization by GRK2 and β-arrestin-2 Overexpression in HEK293 Cells

G protein-coupled receptor 3 (GPR3) is a constitutively active receptor that maintains high 3′-5′-cyclic adenosine monophosphate (cAMP) levels required for meiotic arrest in oocytes and CNS function. Ligand-activated G protein-coupled receptors (GPCRs) signal at the cell surface and are silenced by phosphorylation and β-arrestin recruitment upon endocytosis. Some GPCRs can also signal from endosomes following internalization. Little is known about the localization, signaling, and regulation of constitutively active GPCRs. We demonstrate herein that exogenously-expressed GPR3 localizes to the cell membrane and undergoes internalization in HEK293 cells. Inhibition of endocytosis increased cell surface-localized GPR3 and cAMP levels while overexpression of GPCR-Kinase 2 (GRK2) and β-arrestin-2 decreased cell surface-localized GPR3 and cAMP levels. GRK2 by itself is sufficient to decrease cAMP production but both GRK2 and β-arrestin-2 are required to decrease cell surface GPR3. GRK2 regulates GPR3 independently of its kinase activity since a kinase inactive GRK2-K220R mutant significantly decreased cAMP levels. However, GRK2-K220R and β-arrestin-2 do not diminish cell surface GPR3, suggesting that phosphorylation is required to induce GPR3 internalization. To understand which residues are targeted for desensitization, we mutated potential phosphorylation sites in the third intracellular loop and C-terminus and examined the effect on cAMP and receptor surface localization. Mutation of residues in the third intracellular loop dramatically increased cAMP levels whereas mutation of residues in the C-terminus produced cAMP levels comparable to GPR3 wild type. Interestingly, both mutations significantly reduced cell surface expression of GPR3. These results demonstrate that GPR3 signals at the plasma membrane and can be silenced by GRK2/β-arrestin overexpression. These results also strongly implicate the serine and/or threonine residues in the third intracellular loop in the regulation of GPR3 activity.     

Phosphorylation pattern of the NDUFS4 subunit of complex I of the mammalian respiratory chain

The NDUFS4 subunit of complex I of the mammalian respiratory chain has a fully conserved carboxy-terminus with a canonical RVSTK phosphorylation site. Immunochemical analysis with specific antibodies shows that the serine in this site of the protein is natively present in complex I in both the phosphorylated and non-phosphorylated state. Two-dimensional IEF/SDS–PAGE electrophoresis, ³²P labelling and immunodetection show that “in vitro” PKA phosphorylates the serine in the C-terminus of the NDUFS4 subunit in isolated bovine complex I. ³²P labelling and TLC phosphoaminoacid mapping show that PKA phosphorylates serine and threonine residues in the purified heterologous human NDUFS4 protein.