Oxidative phosphorylation by pyridoxine-deficient rat brain mitochondria

Abstract— Oxygen uptake, ADP/O ratios and respiratory control ratios (RCR) were studied by the oxygen electrode technique in mitochondria prepared from adult and neonatal brains from normal and pyridoxine-deficient rats. The mitochondria from neonatal brain exhibited decreased rates of substrate oxidation, ADP/O ratios and respiratory control ratios in comparison to those obtained with mitochondria from the respective adult brains. The cytochrome contents of the neonatal brains were also less than those of the adults. Within the neonatal or adult groups, there were no differences in any of the parameters tested between the normal and pyridoxine-deficient rats.     

Identification of new tyrosine phosphorylated proteins in rat brain mitochondria

Reversible protein-phosphorylation is emerging as a key player in the regulation of mitochondrial functions. In particular tyrosine phosphorylation represents a promising field to highlight new mechanisms of bioenergetic regulation. Utilizing immunoaffinity enrichment of phosphotyrosine-containing peptides coupled to mass spectrometric analysis we detected new tyrosine phosphorylated proteins in rat brain mitochondria after peroxovanadate treatment. By bioinformatic predictions we provide suggestions about the potential role of tyrosine phosphorylation in mitochondrial physiology. Our results indicate a primary role of tyrosine phosphorylation in regulating energy production at the mitochondrial level. Moreover, tyrosine phosphorylation might regulate the mitochondrial membrane permeability targeting protein complexes containing ADP/ATP translocase, VDAC, creatine kinase and hexokinase.     

Pyk2- and Src-dependent tyrosine phosphorylation of PDK1 regulates focal adhesions

3-Phosphoinositide-dependent protein kinase 1 (PDK1) is a signal integrator that activates the AGC superfamily of serine/threonine kinases. PDK1 is phosphorylated on tyrosine by oxidants, although its regulation by agonists that stimulate G-protein-coupled receptor signaling pathways and the physiological consequences of tyrosine phosphorylation in this setting have not been fully identified. We found that angiotensin II stimulates the tyrosine phosphorylation of PDK1 in vascular smooth muscle in a calcium- and c-Src-dependent manner. The calcium-activated tyrosine kinase Pyk2 acts as a scaffold for Src-dependent phosphorylation of PDK1 on Tyr9, which permits phosphorylation of Tyr373 and -376 by Src. This critical function of Pyk2 is further supported by the observation that Pyk2 and tyrosine-phosphorylated PDK1 colocalize in focal adhesions after angiotensin II stimulation. Importantly, infection of smooth muscle cells with a Tyr9 mutant of PDK1 inhibits angiotensin II-induced tyrosine phosphorylation of paxillin and focal adhesion formation. These observations identify a novel interaction between PDK1 and Pyk2 that regulates the integrity of focal adhesions, which are major compartments for integrating signals for cell growth, apoptosis, and migration.     

Stimulation of tyrosine phosphorylation of rat brain membrane proteins by calmodulin

Calmodulin stimulates the alkali-resistant phosphorylation of peptides of 50 and 58-60 kDa in rat brain membrane. Phosphoamino acid analysis indicated a calmodulin stimulated increase of phosphotyrosine in these peptides. Calmodulin also stimulated the phosphorylation of these peptides at serine and threonine residues. This suggests the involvement of the calmodulin regulatory system in the effects of tyrosine protein kinases.     

Src-dependent tyrosine phosphorylation regulates dynamin self-assembly and ligand-induced endocytosis of the epidermal growth factor receptor

Endocytosis of ligand-activated receptors requires dynamin-mediated GTP hydrolysis, which is regulated by dynamin self-assembly. Here, we demonstrate that phosphorylation of dynamin I by c-Src induces its self-assembly and increases its GTPase activity. Electron microscopic analyses reveal that tyrosine-phosphorylated dynamin I spontaneously self-assembles into large stacks of rings. Tyrosine 597 was identified as being phosphorylated both in vitro and in cultured cells following epidermal growth factor receptor stimulation. The replacement of tyrosine 597 with phenylalanine impairs Src kinase-induced dynamin I self-assembly and GTPase activity in vitro. Expression of Y597F dynamin I in cells attenuates agonist-driven epidermal growth factor receptor internalization. Thus, c-Src-mediated tyrosine phosphorylation is required for the function of dynamin in ligand-induced signaling receptor internalization.     

Protein phosphorylation in rat liver mitochondria

Summary Incubation of rat liver mitochondria in the presence of either [³²P] Pi or ₃₂ ^y -P] ATP resulted in a phosphorylation of four proteins with Mr 50, 47, 44 and 36 kDa, respectively. The endogenous phosphorylation of these proteins in the presence of [³²P] Pi was markedly influenced by the osmolarity of the incubation medium and differentially affected by various effectors of mitochondrial functions, such as Ca²⁺, oligomycin, FCCP, arsenite and dichloroacetate. In particular, the 36 kDa protein, unlike the other proteins, appears to be phosphorylated also by direct incorporation of [³²P], independently of respiratory chain-linked ATP synthesis. The four proteins, located in the mitoplasts, seem to be phosphorylated by diiferent protein kinases, as suggested by the observation that the endogenous phosphorylation of 36 kDa protein resulted selectively increased by addition of exogenous protein kinases, such as casein kinases S and TS. A tentative identification of these phosphorylatable protein is discussed.     

Deoxyadenosine deamination and phosphorylation in rat liver mitochondria

1. Deoxyadenosine metabolism was measured in freshly isolated mitochondria; these organelles took up the deoxynucleoside and formed three detectable products: deoxyinosine, dAMP and dIMP. 2. Enzyme extracts prepared from sonicated mitochondria exhibited deoxyadenosine deaminase, deoxyadenosine kinase, dAMP deaminase and deoxyinosine kinase activities. 3. These data suggest that deoxyadenosine was initially altered in mitochondria by at least two metabolic reactions--deamination and phosphorylation. Deoxyinosine and dAMP were produced. 4. These two products were subsequently phosphorylated and deaminated, respectively to produce dIMP.     

Src-dependent phosphorylation of ASAP1 regulates podosomes

Invadopodia are Src-induced cellular structures that are thought to mediate tumor invasion. ASAP1, an Arf GTPase-activating protein (GAP) containing Src homology 3 (SH3) and Bin, amphiphysin, and RVS161/167 (BAR) domains, is a substrate of Src that controls invadopodia. We have examined the structural requirements for ASAP1-dependent formation of invadopodia and related structures in NIH 3T3 fibroblasts called podosomes. We found that both predominant splice variants of ASAP1 (ASAP1a and ASAP1b) associated with invadopodia and podosomes. Podosomes were highly dynamic, with rapid turnover of both ASAP1 and actin. Reduction of ASAP1 levels by small interfering RNA blocked formation of invadopodia and podosomes. Podosomes were formed in NIH 3T3 fibroblasts in which endogenous ASAP1 was replaced with either recombinant ASAP1a or ASAP1b. ASAP1 mutants that lacked the Src binding site or GAP activity functioned as well as wild-type ASAP1 in the formation of podosomes. Recombinant ASAP1 lacking the BAR domain, the SH3 domain, or the Src phosphorylation site did not support podosome formation. Based on these results, we conclude that ASAP1 is a critical target of tyrosine kinase signaling involved in the regulation of podosomes and invadopodia and speculate that ASAP1 may function as a coincidence detector of simultaneous protein association through the ASAP1 SH3 domain and phosphorylation by Src.     

Characterization of a tyrosine sulfotransferase in rat brain using cholecystokinin derivatives as acceptors

An apparently novel tyrosyl sulfotransferase activity was detected in a crude microsomal fraction from rat cerebral cortex by using 3'-phosphoadenosine 5'-phospho[35S]sulfate [( 35S]PAPS) as the sulfate donor and various cholecystokinin (CCK) fragments or derivatives as acceptors. Among the latter, the shortest substrate was tert-butoxycarbonylaspartyltyrosine (Boc-Asp-Tyr), but the reaction was optimized by increasing the length of the peptide sequence on the C-terminal side up to tert-butoxycarbonylcholecystokinin octapeptide (Boc-CCK-8) as well as by the presence of acidic amino acid residues at the N-terminal side. Peptides with an N-terminal Tyr residue (e.g., CCK-7 or enkephalins) were not sulfated. With Boc-CCK-8 the optimum pH was 5.8, and apparent KM values were 0.14 +/- 0.02 mM for the peptide (0.5 microM PAPS) and 0.12 +/- 0.01 microM for PAPS (0.25 mM Boc-CCK-8). In the presence of 0.2 mM MnCl2 the Vmax of the reaction was enhanced without change of apparent affinities of the two substrates. The possible role of this sulfotransferase activity in posttranslational modification of CCK and other secretory proteins is suggested.     

Src-dependent phosphorylation of the epidermal growth factor receptor on tyrosine 845 is required for zinc-induced Ras activation

Previous studies have shown that exposure of cells to Zn2+ ions induces Ras and MAPK activation through the EGF receptor (EGFR). To further determine the role of EGFR in Zn2+-induced signaling, mouse B82L fibroblasts expressing no detectable EGFR protein (B82L-par), wild type EGFR (B82L-wt), kinase-deficient EGFR (B82L-K721M), or COOH-truncated EGFR (B82L-c'958) were tested. Exposure to Zn2+ induced Ras activity in B82L-wt, B82L-K721M, and B82L-c'958 but not in B82L-par cells, indicating that the tyrosine kinase domain and the auto-phosphorylation sites of the EGFR were not required for Zn2+-induced Ras activation. Zn2+ induced Src activation in all B82L cell lines, including B82L-par, indicating that Src activation is independent of the presence of the EGFR. A Src kinase inhibitor blocked Zn2+-induced Ras activation in all the B82L cell lines capable of this response, suggesting the involvement of Src kinase in Zn2+-induced Ras activation via the EGFR. Zn2+ induced the association of the EGFR with Src and specifically increased the phosphorylation of EGFR at tyrosine 845 (Tyr-845), a known Src phosphorylation site. Stably transfected B82L cells with a point mutation of the EGFR at Tyr-845 (B82L-Y845F) exhibited only basal Ras activity following exposure to Zn2+. These data demonstrate that Src-dependent phosphorylation of the EGFR at Tyr-845 is required for EGFR transactivation and Zn2+-induced Ras activation.     

Circadian rhythms of oxidative phosphorylation: effects of rotenone and melatonin on isolated rat brain mitochondria

Mitochondrial experiments are of increasing interest in different fields of research. Inhibition of mitochondrian activities seems to play a role in Parkinson's disease and in this regard several animal models have used inhibitors of mitochondrial respiration such as rotenone or MPTP. Most of these experiments were done during the daytime. However, there is no reason for mitochondrial respiration to be constant during the 24h. This study investigated the circadian variation of oxidative phosphorylation in isolated rat brain mitochondria and the administration-time-dependent effect of rotenone and melatonin. The respiratory control ratio, state 3 and state 4, displayed a circadian fluctuation. The highest respiratory control ratio value (3.01) occurred at 04:00h, and the lowest value (2.63) at 08:00h. The highest value of state 3 and state 4 oxidative respiration occurred at 12:00h and the lowest one at 20:00h. The 24h mean decrease in the respiratory control ratio following incubation with melatonin and rotenone was 7 and 32%, respectively; however, the exact amount of the inhibition exerted by these agents varied according to the time of the mitochondria isolation. Our results show the time of mitochondrial isolation could lead to interindividual variability. When studies require mitochondrial isolation from several animals, the time between animal experiments has to be minimized. In oxidative phosphorylation studies, the time of mitochondria isolation must be taken into account, or at least specified in the methods section.     

Src-dependent phosphorylation of membrane type I matrix metalloproteinase on cytoplasmic tyrosine 573: role in endothelial and tumor cell migration

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a transmembrane MMP that plays important roles in migratory processes underlying tumor invasion and angiogenesis. In addition to its matrix degrading activity, MT1-MMP also contains a short cytoplasmic domain whose involvement in cell locomotion seems important but remains poorly understood. In this study, we show that MT1-MMP is phosphorylated on the unique tyrosine residue located within this cytoplasmic sequence (Tyr(573)) and that this phosphorylation requires the kinase Src. Using phosphospecific antibodies recognizing MT1-MMP phosphorylated on Tyr(573), we observed that tyrosine phosphorylation of the enzyme is rapidly induced upon stimulation of tumor and endothelial cells with the platelet-derived chemoattractant sphingosine-1-phosphate, suggesting a role in migration triggered by this lysophospholipid. Accordingly, overexpression of a nonphosphorylable MT1-MMP mutant (Y573F) blocked sphingosine-1-phosphate-induced migration of Human umbilical vein endothelial cells and HT-1080 (human fibrosarcoma) cells and failed to stimulate migration of cells lacking the enzyme (bovine aortic endothelial cells). Altogether, these findings strongly suggest that the Src-dependent tyrosine phosphorylation of MT1-MMP plays a key role in cell migration and further emphasize the importance of the cytoplasmic domain of the enzyme in this process.