Mitochondria constantly respond to changes in substrate availability and energy utilization to maintain cellular ATP supplies, and at the same time control reactive oxygen radical (ROS) production. Reversible phosphorylation of mitochondrial proteins has been proposed to play a fundamental role in metabolic homeostasis, but very little is known about the signalling pathways involved. We show here that Protein Kinase A (PKA) regulates ATP production by phosphorylation of mitochondrial proteins, including subunits of cytochrome c oxidase. The cyclic AMP (cAMP) which activates mitochondrial PKA does not originate from cytoplasmic sources but is generated within mitochondria by the carbon dioxide/bicarbonate-regulated soluble adenylyl cyclase (sAC) in response to metabolically generated carbon dioxide. We demonstrate for the first time the existence of a CO2-HCO3−-sAC-cAMP-PKA (mito-sAC) signalling cascade wholly contained within mitochondria, which serves as a metabolic sensor modulating ATP generation and ROS production in response to nutrient availability.

Mammalian fertilization is dependent upon a series of bicarbonate-induced, cAMP-dependent processes sperm undergo as they "capacitate," i.e., acquire the ability to fertilize eggs. Male mice lacking the bicarbonate- and calcium-responsive soluble adenylyl cyclase (sAC), the predominant source of cAMP in male germ cells, are infertile, as the sperm are immotile. Membrane-permeable cAMP analogs are reported to rescue the motility defect, but we now show that these "rescued" null sperm were not hyperactive, displayed flagellar angulation, and remained unable to fertilize eggs in vitro. These deficits uncover a requirement for sAC during spermatogenesis and/or epididymal maturation and reveal limitations inherent in studying sAC function using knockout mice. To circumvent this restriction, we identified a specific sAC inhibitor that allowed temporal control over sAC activity. This inhibitor revealed that capacitation is defined by separable events: induction of protein tyrosine phosphorylation and motility are sAC dependent while acrosomal exocytosis is not dependent on sAC.

The second messenger cAMP has been extensively studied for half a century, but the plethora of regulatory mechanisms controlling cAMP synthesis in mammalian cells is just beginning to be revealed. In mammalian cells, cAMP is produced by two evolutionary related families of adenylyl cyclases, soluble adenylyl cyclases (sAC) and transmembrane adenylyl cyclases (tmAC). These two enzyme families serve distinct physiological functions. They share a conserved overall architecture in their catalytic domains and a common catalytic mechanism, but they differ in their sub-cellular localizations and responses to various regulators. The major regulators of tmACs are heterotrimeric G proteins, which transduce extracellular signals via G protein-coupled receptors. sAC enzymes, in contrast, are regulated by the intracellular signaling molecules bicarbonate and calcium. Here, we discuss and compare the biochemical, structural and regulatory characteristics of the two mammalian AC families. This comparison reveals the mechanisms underlying their different properties but also illustrates many unifying themes for these evolutionary related signaling enzymes.

Intracellular targets of the ubiquitous second messenger cAMP are located at great distances from the most widely studied source of cAMP, the G protein responsive transmembrane adenylyl cyclases. We previously identified an alternative source of cAMP in mammalian cells lacking transmembrane spanning domains, the "soluble" adenylyl cyclase (sAC). We now demonstrate that sAC is distributed in specific subcellular compartments: mitochondria, centrioles, mitotic spindles, mid-bodies, and nuclei, all of which contain cAMP targets. Distribution at these intracellular sites proves that adenylyl cyclases are in close proximity to all cAMP effectors, suggesting a model in which local concentrations of cAMP are regulated by individual adenylyl cyclases targeted to specific microdomains throughout the cell.

Bicarbonate-responsive “soluble” adenylyl cyclase resides, in part, inside the mammalian cell nucleus where it stimulates the activity of nuclear protein kinase A to phosphorylate the cAMP response element binding protein (CREB). The existence of this complete and functional, nuclear-localized cAMP pathway establishes that cAMP signals in intracellular microdomains and identifies an alternate pathway leading to CREB activation.