Analysis of Steady-state Protein Phosphorylation in Mitochondria Using a Novel Fluorescent Phosphosensor Dye

Abstract

The phosphorylation of mitochondrial proteins is pivotal to the regulation of respiratory activity in the cell and to signaling pathways leading to apoptosis, as well as for other vital mitochondrial processes. A number of protein kinases have been identified in mitochondria but the physiological substrates for many of these remain unknown or poorly understood. By necessity, most studies of mitochondrial phosphoproteins to date have been conducted using in vitro incorporation of 32 P. However, proteins that are highly phosphorylated from in situ reactions are not necessarily detected by this approach. In this study, a new small molecule fluorophore has been employed to characterize steady-state levels of mitochondrial phosphoproteins. The dye is capable of sensitive detection of phosphorylated amino acid residues in proteins separated by gel electrophoresis. When the fluorescent dye is combined with a total protein stain in a sequential gel staining procedure, the phosphorylated proteins can be visualized in the same gel as the total proteins. To optimize resolution of the proteins in mitochondria, a previously described sucrose gradient fractionation method was employed prior to gel electrophoresis. Phosphorylated proteins, as defined by the fluorescence of the phosphosensor, were excised from the gels and identified by peptide mass fingerprinting. One novel and prominent phosphoprotein identified in this manner was determined to be the 42-kDa subunit of mitochondrial complex I.A number of protein kinases are known to be localized within mitochondria, including pyruvate dehydrogenase kinase, branched-chain ␣-ketoacid dehydrogenase kinase, cAMP-dependent protein kinase, protein kinase C␦, stress-activated protein kinase, and A-Raf, as well as an unidentified tyrosine kinase (1, 2). Determination of the physiological substrates of many of these kinases has proved to be challenging. Global analysis of mitochondrial phosphoproteins has to date been performed by incubating isolated mitochondria with [␥-2 P]ATP (2-9) or by labeling cells cultured in phosphate-free medium with [32 P]orthophosphate (10). While such methods provide information concerning the capability of these proteins to be phosphorylated during the actual time period of radiolabeling, they do not identify proteins already phosphorylated to significant levels prior to the labeling step. In addition, metabolically incorporating radiolabels, using standard doses and time courses, have previously been shown to induce DNA fragmentation, elevate p53 tumor suppressor protein levels, alter cell/ nuclear morphology, and result in cell cycle arrest or apoptosis (11)(12)(13)(14). Unlike radiolabeling, Western blotting potentially identifies all the phosphorylated proteins in samples (2, 8). However, no broad-spectrum phosphoamino acid-reactive antibodies exist, and although high quality antibodies to phosphotyrosine residues are available, antibodies that specifically recognize phosphoserine and phosphothreonine residues are typically sensitive to amino acid se...

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