Localization of PTP-1B, SHP-2, and Src exclusively in rat brain mitochondria and functional consequences

Abstract

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.

FIGURE 1.

PTP-1B, SHP-2, and Src expression in mitochondria from different rat tissues and human platelets. The protein content in purified mitochondria from the different tissues was assayed by the Micro-BCA method, and the same amount of protein lysate was loaded per lane: 100 μg for immunodetection of PTP-1B, SHP-2, and GRP78 and 30 μg for Src and manganese superoxide dismutase (MnSOD). For platelets, 10 μg of proteins were used.

FIGURE 2.

Immunogold labeling of PTP-1B in isolated brain mitochondria. An overall view and a mitochondrium was shown, in which PTP-1B (A and B) and subunit I of complex IV (C) were immunodetected with 15- or 10-nm gold-labeled secondary antibody.

FIGURE 3.

Changes in tyrosine phosphorylation of mitochondrial proteins from liver, kidney, heart, and muscle. Mitochondria (20 μg) were untreated (lane 1) or treated at 30 °C for 10 min with 1 mm ATP alone (lane 2) or after preincubation at 30 °C for 10 min with 10 μm PP2 (lane 3) or 2 mm orthovanadate plus 10 mm NaF (lane 4). The reaction was stopped by the addition of 5× Laemmli buffer. Phosphorylated proteins on tyrosine residues were detected with an antibody to phosphotyrosine directly labeled with horseradish peroxidase. The membranes were stripped and reprobed with an antibody to manganese superoxide dismutase (MnSOD) as a loading control (below). Data are representative of at last three experiments.

FIGURE 4.

Changes in tyrosine phosphorylation of mitochondrial proteins from brain. Left, see the legend for Fig. 3. Right, similar to left, except that 10 and 100 μm PP1 were used. MnSOD, manganese superoxide dismutase.

FIGURE 5.

Phosphorylation status of Src in brain mitochondria. Left, Src in untreated or ATP-treated mitochondria (20 μg) was immunodetected with the anti pY416-Src antibody. The membrane was stripped and reprobed with the anti non-pY416-Src antibody, stripped again, and reprobed with an anti Src antibody. Right, a similar procedure was used to study Src phosphorylation at Tyr-527 residue.