Mitochondrial AKAP121 Binds and Targets Protein Tyrosine Phosphatase D1, a Novel Positive Regulator of src Signaling

An immunodetection study of protein tyrosine phosphatase 1B (PTP-1B), SHP-2, and Src in isolated mitochondria from different rat tissues (brain, muscle, heart, liver, and kidney) revealed their exclusive localization in the brain. Given this result, we sought whether mitochondria respond to ATP and to the general tyrosine phosphatase inhibitor orthovanadate and found little or no change in the tyrosine phosphorylation profile of mitochondria from muscle, heart, liver, and kidney. In contrast, ATP induced an enhancement in the tyrosine-phosphorylated protein profile of brain mitochondria, which was further greatly enhanced with orthovanadate and which disappeared when Src was inhibited with two inhibitors: PP2 and PP1. Importantly, we found that in brain mitochondria, ATP addition induced Src autophosphorylation at Tyr-416 in its catalytic site, leading to its activation, whereas the regulatory Tyr-527 site remained unphosphorylated. Functional implications were addressed by measurements of the enzymatic activity of each of the oxidative phosphorylation complexes in brain mitochondria in the presence of ATP. We found an increase in complex I, III, and IV activity and a decrease in complex V activity, partially reversed by Src inhibition, demonstrating that the complexes are Src substrates. These results complemented and reinforced our initial study showing that respiration of brain mitochondria was partially dependent on tyrosine phosphorylation. Therefore, the present data suggest a possible control point in the regulation of respiration by tyrosine phosphorylation of the complexes mediated by Src auto-activation.Mitochondria provide the energy necessary for cell growth and biological activities through oxidative phosphorylation (OxPhos). 4 This relies on electron transfer from oxidative substrates to oxygen, via a series of redox reactions, to generate water. In this process, protons are pumped from the matrix across the mitochondrial inner membrane, via respiratory complexes I, III, and IV. When protons return to the mitochondrial matrix, ATP is synthesized via complex V.As the energy demand of a cell depends on its function and activity, energy production is adjusted and controlled by different mechanisms (1-3). One major regulatory system is protein phosphorylation/dephosphorylation (4). Evidence indicates that mitochondrial proteins undergo posttranslational phosphorylation (2, 5, 6), and reports have unambiguously revealed the existence of several kinases within the mitochondria, such as cAMP-dependent protein kinase (7) and members of the Src kinase family (8). Protein phosphatases have also been described, such as Ser/Thr phosphatases, PP2C-␥ and PP2A (7), and tyrosine phosphatases, SHP-2 (9), PTP-1B (10), and PTPMT1, a dual-specific phosphatase (11), the latter found exclusively in mitochondria but not in the cytosol.As indicated by earlier reports, mitochondrial signaling enzyme distribution and stimulation may vary according to the tissue. In a preceding report, which also explored the presence of PTP...

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Localization of PTP-1B, SHP-2, and Src Exclusively in Rat Brain Mitochondria and Functional Consequences

Arachiche

Augereau

Décossas

et al. 2008

“…HA epitope was placed at the extreme NH 2 terminus of PTPD1 by PCR (22). The HAtagged PTPD1 mutants were generated by PCR using the following primers: PTPD1 ⌬1-581 forward, 5Ј-cggggtaccGTCCCG-CCACCTTTACATCAGC (KpnI in lowercase type); reverse, 5Ј-tgctctagaGCAGATGAGCCTGGAGCTTTTCAG (XbaI in lowercase type); PTPD1 ⌬1-325 forward, 5Ј-ggggtaccaTCTTCA-AGGATGTCTCTGCCT (KpnI in lowercase type).…”

Section: Methodsmentioning

confidence: 99%

“…The PTPD1-Src-AKAP121 complex is required for efficient maintenance of mitochondrial membrane potential and ATP oxidative synthesis. When not in complex with AKAP121, PTPD1 directs EGF-Src signaling to the nucleus, activating ERK1/2-and Elk1-dependent gene transcription (22)(23)(24).…”

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confidence: 99%

Protein-tyrosine Phosphatase PTPD1 Regulates Focal Adhesion Kinase Autophosphorylation and Cell Migration

Carlucci

Gedressi

Lignitto

et al. 2008

Self Cite

PTPD1 is a cytosolic nonreceptor tyrosine phosphatase and a positive regulator of the Src-epidermal growth factor transduction pathway. We show that PTPD1 localizes along actin filaments and at adhesion plaques. PTPD1 forms a stable complex via distinct molecular modules with actin, Src tyrosine kinase, and focal adhesion kinase (FAK), a scaffold protein kinase enriched at adhesion plaques. Overexpression of PTPD1 promoted cell scattering and migration, short hairpin RNA-mediated silencing of endogenous PTPD1, or expression of PTPD1 mutants lacking either catalytic activity (PTPD1 C1108S ) or the FERM domain (PTPD1 ⌬1-325 ) significantly reduced cell motility. PTPD1 and Src catalytic activities were both required for epidermal growth factor-induced FAK autophosphorylation at its active site and for downstream propagation of ERK1/2 signaling. Our findings demonstrate that PTPD1 is a component of a multivalent scaffold complex nucleated by FAK at specific intracellular sites. By modulating Src-FAK signaling at adhesion sites, PTPD1 promotes the cytoskeleton events that induce cell adhesion and migration.

“…However, central for PKA-mediated phosphorylation under physiological conditions are protein kinase A-anchoring proteins (AKAPs), which localize PKA to cellular compartments in close proximity to its targets (44); S-AKAP84 and AKAP121 have been shown to mediate localization of PKA to the outer side of the outer mitochondrial membrane and to be crucial for cAMP-dependent signaling to the mitochondria (45)(46)(47). Also, in this study we found that incubation of isolated mitochondria with cAMP did not change COX activity.…”

Section: Fig 2 Nano-liquid Chromatography/electrospray Ionization/ mentioning

confidence: 99%

cAMP-dependent Tyrosine Phosphorylation of Subunit I Inhibits Cytochrome c Oxidase Activity

Lee

Salomon

Ficarro

et al. 2005

188

Signaling pathways targeting mitochondria are poorly understood. We here examine phosphorylation by the cAMP-dependent pathway of subunits of cytochrome c oxidase (COX), the terminal enzyme of the electron transport chain. Using anti-phospho antibodies, we show that cow liver COX subunit I is tyrosinephosphorylated in the presence of theophylline, a phosphodiesterase inhibitor that creates high cAMP levels, but not in its absence. The site of phosphorylation, identified by mass spectrometry, is tyrosine 304 of COX catalytic subunit I. Subunit I phosphorylation leads to a decrease of V max and an increase of K m for cytochrome c and shifts the reaction kinetics from hyperbolic to sigmoidal such that COX is fully or strongly inhibited up to 10 M cytochrome c substrate concentrations, even in the presence of allosteric activator ADP. To assess our findings with the isolated enzyme in a physiological context, we tested the starvation signal glucagon on human HepG2 cells and cow liver tissue. Glucagon leads to COX inactivation, an effect also observed after incubation with adenylyl cyclase activator forskolin. Thus, the glucagon receptor/G-protein/cAMP pathway regulates COX activity. At therapeutic concentrations used for asthma relief, theophylline causes lung COX inhibition and decreases cellular ATP levels, suggesting a mechanism for its clinical action.Cytochrome c oxidase (COX), 1 the terminal enzyme of the mitochondrial respiratory chain, reduces oxygen to water and pumps protons across the inner mitochondrial membrane. COX contains 13 subunits per monomer, three of which are encoded by the mitochondrial genome. COX has been shown to be the rate-limiting enzyme of oxidative metabolism under physiological conditions in a variety of human cell types (1) and in a mouse cell line with a mutation in COX subunit I (2). The functional mammalian enzyme has been crystallized as a dimer (3) and shows three features, described below, that are found in key metabolic enzymes: allosteric regulation, isoforms, and phosphorylation. COX activity is regulated by small molecules such as ATP and ADP (4 -6), and the thyroid hormone T2 (7). COX contains skeletal muscle/heart ("heart-type") and nonskeletal muscle ("liver-type") isoforms of subunits VIa, VIIa, and VIII that have been known for the past 2 decades (reviewed in Ref. 8). We have recently discovered three additional isoforms: a lung-specific isoform of subunit IV, a third isoform of subunit VIII, and a testes-specific isoform of subunit VIb (9 -11). Although it is clear that protein kinases and phosphatases are crucial in cellular signaling, little is known about their role in the regulation of the respiratory chain complexes.There have been three studies of COX phosphorylation: Steenaart and Shore (12) examined mitochondrial proteins phosphorylated by endogenous kinases in the presence of [␥-32 P]ATP and identified COX subunit IV; Miyazaki et al. (13) showed that COX subunit II can be phosphorylated by nonreceptor tyrosine kinase c-Src in osteoblasts and found a positive ...