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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 15, 2002 as doi:10.1096/fj.02-0598fje. |
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,2
,2,3
* Department of Pharmacology,
Tri-Institutional MD/PhD Program,
Department of Cell Biology, Joan and Sanford I. Weill Medical College of Cornell University, New York, New York, USA
5Correspondence: Department of Pharmacology, Weill Medical College of Cornell University, 1300 York Ave., New York, NY 10021, USA. E-mail: llevin{at}med.cornell.edu
SPECIFIC AIMS
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 (tmACs). We examined whether the "soluble" adenylyl cyclase (sAC), an alternative source of cAMP in mammalian cells lacking transmembrane spanning domains, was localized near the intracellular targets of cAMP.
PRINCIPAL FINDINGS
1. sAC is localized to the nucleus
To determine the subcellular distribution of sAC, we generated three affinity-purified polyclonal antisera to distinct, nonoverlapping portions of sAC protein (
N-term, Middle, and C-term) and a panel of monoclonal antibodies to the amino-terminal catalytic domains. The N-term polyclonal antisera and the panel of monoclonal antibodies recognized full-length sAC protein (187 kDa) (sACfl) and a 50 kDa truncated isoform (sACt) generated by an alternatively spliced transcript, whereas Middle and C-term antibodies recognized only the full-length isoform.
We observed positive immunoreactivity in COS7 cells with each antibody in the nuclei and both diffuse and particulate staining of the cytoplasm (Fig. 1
). Qualitatively similar staining patterns were observed with each antibody in all cell lines and primary cells, including a variety of mammalian transformed cell lines MDCK, HeLa, HEK293, Hepa-1, PC12, SHSY-5Y, and TM4 as well as immortalized (NIH3T3) and primary fibroblasts. Confocal microscopy and thin optical sectioning indicated that sAC protein was present inside the nucleus. Immunocytochemistry using Middle (data not shown) and C-term (Fig. 1C, D
) antisera indicated sACfl was in the nucleus; Western analysis confirmed the nuclear presence of sACt.
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2. sAC is localized to mitochondria
The punctate cytoplasmic sAC immunostaining (Fig. 1A, C, E, F
) colocalized with antibodies to cytochrome c oxidase (COX) (data not shown) and the mitochondrial marker "Mitotracker" (Fig. 1B, D
). Mitochondrial localization of sAC was confirmed by Western analysis of mitochondrial fractions isolated from rat liver using a Percoll density gradient.
3. sAC is localized to microtubules and centrioles
A juxtanuclear cap corresponding to the centrosome contained diffuse sAC staining (Fig. 1)
and one or two bright spots interpreted to be centrioles. These spots were at the center of the microtubule network and were unaffected by the microtubule depolymerizer nocodazole. Their identification as centrioles was confirmed by sAC immunolocalization through mitosis during which the centrioles were positioned at the poles of the spindle apparatus. During prophase, sAC dispersed from the nucleus (data not shown) and, in metaphase, accumulated at the mitotic poles and spindle fibers (Fig. 2
A–C). It remained associated with the poles during anaphase (Fig. 2D-F
). During cytokinesis, centriolar staining was no longer apparent, but the midbody was strongly immunoreactive (Fig. 2I
, inset). At all stages of mitosis, the condensed chromosomes were negative for sAC staining (Fig. 2C, F
). After cytokinesis, sAC resumed its localization within the nucleus (Fig. 2G-I
).
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4. sAC in organelles is functional
Bicarbonate elicited an eightfold increase in cAMP in nuclear extracts whereas forskolin produced no significant change. Thus, sAC protein in nuclei is active and appears to be the only functional form of adenylyl cyclase present. Mitochondrial sAC is also functional; sAC protein is present and cAMP is generated in response to bicarbonate in isolated mitochondria.
CONCLUSIONS AND SIGNIFICANCE
In mammals, the ubiquitous second messenger cAMP is synthesized by two classes of adenylyl cyclase (AC): the G-protein-responsive transmembrane adenylyl cyclases (tmACs) and the widely distributed, bicarbonate-responsive ‘soluble’ adenylyl cyclase (sAC). cAMP mediates its cellular effects via at least three distinct classes of direct effectors: cAMP-dependent protein kinase (PKA), RAP exchange proteins, and cAMP gated ion channels (cNGC). cNGCs and a subset of PKA targets are localized to the plasma membrane, near tmACs, in what appear to be macromolecular signaling complexes comprised of the G-protein coupled receptor, G-protein, tmAC, PKA, and its ultimate substrate. PKAs are also tethered throughout the cytoplasm to a variety of intracellular locations distant from the plasma membrane by a family of scaffolding proteins termed A kinase-anchoring proteins (AKAPs). For cAMP generated by tmACs to activate these intracellular targets, the second messenger would have to diffuse over long distances within the cell. Second messenger diffusion would diminish specificity and selectivity of signaling and would be inefficient considering the cytoplasm contains many cAMP-catabolizing phosphodiesterases. Recent methods for quantifying cAMP generation by G-protein-regulated tmACs reveal that cAMP diffusion from the plasma membrane is restricted to <1 µm. These data suggest there must be a source of cAMP in close proximity to its intracellular effectors.
Bicarbonate-regulated sAC was originally purified from testis cytosol but has since been found in a wide variety of tissues. It represents an alternative source for cAMP that we now show, contrary to its name, is not solely a soluble protein but is specifically targeted to well-defined intracellular compartments—mitochondria, centrioles, mitotic spindles, midbodies, and nuclei—in close proximity to intracellular effectors of cAMP signaling. Distribution at these intracellular sites proves that adenylyl cyclases are in close proximity to cAMP effectors, supporting a model in which local concentrations of cAMP are regulated by individual adenylyl cyclases targeted to specific microdomains throughout the cell (Fig. 3
).
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Localization of sAC inside the nucleus is surprising. It is widely accepted that cAMP functions to regulate gene expression, and immunolocalization studies have revealed PKA catalytic and regulatory subunits in the nucleus. But it had been thought that the direct target of cAMP, PKA, is activated in the cytoplasm and liberated catalytic subunit translocates into the nucleus to phosphorylate cAMP response element binding proteins. Our discovery of a source of cAMP inside the nucleus raises the possibility that the entire signaling cascade also resides within the nucleus (Fig. 3A
).
The Rap1 exchange protein activated by cAMP (Epac) is found at the mitochondria, but its role there has yet to be determined. Close proximity to the intracellular cAMP effector mitochondrial AKAP-tethered PKA suggests that sAC may be the source of cAMP mediating PKA-dependent phosphorylation of the proapoptotic BAD protein and/or PKA mediated inhibition of COX activity. Cellular levels of bicarbonate will reflect changes in CO2, the final metabolite of energy-producing metabolic processes. Therefore, modulation of the electron transport chain by bicarbonate-sensitive sAC provides a possible mechanism for metabolic feedback regulation.
Some AKAPs tether PKA to the microtubule organizing center or centrioles. Centriolar PKA is required for the phosphorylation of centrin preceding spindle formation. The presence of sAC at the centrioles suggests it is the source of cAMP that directs centriolar separation and assembly of the mitotic spindle.
During metaphase, sAC staining is observed at the centrioles and microtubule spindle fibers. At the centrioles, sAC-generated cAMP could initiate a phosphorylation cascade by activating AKAP-tethered PKA, whereas at the microtubules the Rap1 exchange protein Epac is the only identified target of cAMP (Fig. 3B
). Likewise, Epac localization to the midbody during cytokinesis suggests that sAC-generated cAMP regulates the activity of Rap1 important for cell separation (Fig. 3C
).
Due to the ubiquitous presence of carbonic anhydrases, cellular levels of bicarbonate will reflect changes in intracellular pH as well as the metabolite CO2. Therefore, bicarbonate-regulated sAC could be involved in the variety of cellular and physiological processes attributed to each of these modulators, including fluid secretion, acid-base changes, breathing rate, and metabolic feedback control. Contrary to tmAC microdomains which respond to extracellular signals such as hormones and neurotransmitters, sAC-defined microdomains will respond to intrinsic cellular signals, confirming that sAC and tmACs define distinct cAMP signaling systems within mammalian cells.
Finally, organization into discrete microdomains provides a mechanism whereby cAMP could simultaneously participate in a multitude of seemingly disparate functions within a single cell.
FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.02-0598fje; to cite this article, use FASEB J. (November 15, 2002) 10.1096/fj.02-0598fje 
2 These authors contributed equally.
3 Present address: Laboratory of Muscle Biology, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, 50 South Dr., Room 1150, Bethesda, MD 20892-8024, USA.
4 Present address: Cognia Corporation, 117 E. 55th St., New York, NY 10021, USA.
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