Maize Genetics Cooperation Newsletter 80. 2006.

Inhibitory analysis of protein phosphorylation/dephosphory-lation in mitochondria

--Subota, IY; Arziev, AS; Tarasenko, VI; Konstantinov, YM

Phosphorylation/dephosphorylation of amino acid residues is the most common posttranslational modification of proteins which can alter enzyme activity, half-life of the protein, etc. Protein kinases and protein phosphatases which perform this reactions probably function as tightly associated complexes regulated by feedback mechanism. The excess of protein kinase leads to an activation of protein phosphatase which in turn dephosphorylates and inactivates protein kinase.

In spite of the importance of this modification, little is known about the role of protein phosphorylation in plant mitochondria. In this work, we studied the influence of redox conditions on the level of phosphorylation of individual mitochondrial proteins as a possible mechanism of redox regulation of mitochondrial processes, including gene expression.

The mitochondria were isolated from 3-day-old etiolated maize seedlings (hybrid VIR42MV) by the standard method of differential centrifugation. Protein phosphorylation assays were carried out according to Struglics et al. (FEBS Lett. 475:213-217, 2000) with the use of [γ³²P]ATP (specific radioactivity was 6000 Ci/mmol).

Incubation of mitochondria with [γ³²P]ATP resulted in phosphorylation of 8 proteins as judged by SDS-PAGE and autoradiography. Potassium ferricyanide used as an oxidizing agent decreased ³²P incorporation into all of these proteins (Fig. 1). Treatment with the reducing agent sodium dithionite reduced specifically the labeling of the 62 kDa protein. When the physiological redox agent glutathione was used, we also observed some alterations in protein phosphorylation (Fig. 1). The addition of oxidized glutathione

Figure 1. The effect of redox agents and protein kinases and protein phosphatase inhibitors on mitochondrial phosphorylation. Lane 1, no agents; lane 2, 5 mM potassium ferricyanide; lane 3, 5 mM sodium dithionite; lane 4, 5 mM GSH; lane 5, 10 mM GSSG; lane 6, 200 nM staurosporine; lane 7, 40 mM NaF; lane 8, ferricyanide+NaF; lane 9, ferricyanide+staurosporine; lane 10, dithionite+NaF; lane 11, dithionite+staurosporine.

resulted in a substantial decrease in protein phosphorylation. When comparing the inhibitory effects of two oxidising agents on protein phosphorylation, GSSG has been demonstrated to be more effective than potassium ferricyanide. GSH used as a reducing agent inhibited ³²P incorporation into two proteins of 62 and 55 kDa.

In our experiments, 200 nM staurosporine reduced the level of phosphorylation of the 55 kDa protein. Simultaneous addition of redox agents and inhibitors of protein kinases (staurosporine) and protein phosphatases (sodium fluoride) modulated the activity of protein phosphorylation. When potassium ferricyanide was added together with sodium fluoride, the rate of protein phosphorylation increased in comparison with the effects of these agents alone.

Figure 2. Coomassie-stained gel identical to that used in Figure 1. M-ladder; lanes 1 – 11 are the same as in Figure 1.

The Coomassie-stained gel showed equal loading of all lanes (Fig. 2). A comparison with the autoradiograph demonstrated that the polypeptide and phosphoprotein patterns were quite different.

Thus, on the basis of the results obtained, we may conclude that inhibition of both protein kinases and protein phosphatases in different redox conditions clearly affects the protein phosphorylation activity in mitochondria. These data permit a supposition about the existence of redox sensitive protein kinases and protein phosphatases in plant mitochondria which modify the effects of redox conditions on protein phosphorylation in organello.

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