The discovery of a signaling system through sAC and PKA (Acin-Perez et al., 2009b
) contributed to our understanding of the regulation of mitochondrial metabolism. However, the molecular mechanisms underlying this regulation had not been defined.
Many proteins involved in OXPHOS function are targets of phosphorylation (Balaban; Deng et al.; Hopper et al., 2006
). In this study, we focused on COX, but it is likely that other OXPHOS components may be involved in PKA-mediated metabolic regulation (Papa et al., 2008
). COX contains 13 subunits, 10 of which are phosphorylated at various sites (Thomson, 2002
). With a combined proteomic (Acin-Perez et al., 2009b
) and informatics strategy we determined that S58 in COXIV-1 fulfilled the criteria for being accessible to matrix PKA and part of a PKA target motif. Furthermore, phosphorylation of this residue had been shown earlier in bovine heart mitochondria (Helling et al., 2008
). We pinpointed S58 of COXIV-1, as a player in COX regulation by reversible phosphorylation and identified S58 as part of the allosteric binding site for ATP on the matrix side of COXIV-1, showing that phosphorylation of S58 dramatically weakens the interaction with ATP. Furthermore, we have shown that lack of COXIV-1 phosphorylation in S58A mutant cells results in reduced COX activity and defective growth under oxidative conditions. These results suggest a link between activation of the intramitochondrial CO2
-cAMP-PKA pathway and phosphorylation of COXIV-1 S58, which regulates COX allosteric inhibition by matrix ATP. This interpretation does not exclude that extramitochondrial ATP may bind other sites in COX and regulate its activity differently.
Our findings suggest that mitochondria can switch from a “storage mode” to a “consumption mode”. When ATP builds up in mitochondria, because of reduced requirements, enzymes are inhibited and fuel utilization diminishes, resulting in low intramitochondrial CO2 levels, low sAC activity and cAMP, and low PKA activity. COXIV-1 is dephosphorylated and COX activity inhibited by ATP, shunting substrates towards fat and glycogen accumulation. On the other hand, when cellular ATP consumption is high, large amounts of substrates are oxidized, CO2 stimulates COXIV-1 phosphorylation, transiently preventing ATP inhibition and allowing for maximal electron flux through COX and high ATP production. Since the phospho-mimetic S58D results in a small increase in COX activity and the S58phospho-COXIV-1 antibodies immuno-capture most COXIV-1, we inferred that normally the majority of COXIV-1 is phosphorylated. Studies in more complex organisms will assess whether this applies to organs and tissues in vivo.
The S58 residue is conserved among mammals (Supplementary Table 2
), but not in non-mammalian species, suggesting that S58 has coevolved with mammals. Furthermore, two different isoforms of COXIV are found in many species. Similarly to other nuclear-encoded COX subunits, COXIV plays a regulatory role, in part through differential temporal and spatial expression of its isoforms. COXIV-2 does not have a PKA phosphorylation site corresponding to S58. Instead, it has three glutamic acids, carrying negative charges that may impede ATP binding. COXIV-2 confers higher activity to COX than COXIV-1, thereby allowing for a fast electron flux and possibly reducing the risk of forming reactive oxygen species in highly oxygenated tissues (Huttemann et al., 2007
). Under oxygen deprivation COXIV-2 is induced in brain, where it abolishes sensitivity to ATP inhibition (Horvat et al., 2006
). Furthermore, an increase of COXIV-2 expression regulated by hypoxia-inducible factor 1 occurs under hypoxia, in association with COXIV-1 degradation (Fukuda et al., 2007
). These responses could be part of a hypoxia-preconditioning program to prevent the wave of free radical production, following reperfusion.
The presence of S58 in COXIV-1 of mammalian species could be related to a specific need for metabolic regulation in animals exposed to variations in ambient temperature and food availability. In modern days, metabolic regulation through reversible phosphorylation of respiratory chain subunits may represent a trigger for metabolic diseases, such as obesity and diabetes, or a potential target for therapy with approaches to modulate consumption and storage of metabolic supplies.