The cyclic AMP (cAMP) signalling cascade is necessary for cell homeostasis and plays important roles in many processes. This is particularly relevant during ageing and age-related diseases, where drastic changes, generally decreases, in cAMP levels have been associated with the progressive decline in overall cell function and, eventually, the loss of cellular integrity. The functional relevance of reduced cAMP is clearly supported by the finding that increases in cAMP levels can reverse some of the effects of ageing. Nevertheless, despite these observations, the molecular mechanisms underlying the dysregulation of cAMP signalling in ageing are not well understood. Compartmentalization is widely accepted as the modality through which cAMP achieves its functional specificity; therefore, it is important to understand whether and how this mechanism is affected during ageing and to define which is its contribution to this process. Several animal models demonstrate the importance of specific cAMP signalling components in ageing, however, how age-related changes in each of these elements affect the compartmentalization of the cAMP pathway is largely unknown. In this review, we explore the connection of single components of the cAMP signalling cascade to ageing and age-related diseases whilst elaborating the literature in the context of cAMP signalling compartmentalization.

The second messenger cyclic AMP regulates many nuclear processes including transcription, pre-mRNA splicing and mitosis. While most functions are attributed to protein kinase A, accumulating evidence suggests that not all nuclear cyclic AMP-dependent effects are mediated by this kinase, implying that other effectors may be involved. Here we explore the nuclear roles of Exchange Protein Activated by cyclic AMP 1. We find that it enters the nucleus where forms reversible biomolecular condensates in response to cyclic AMP. This phenomenon depends on intrinsically disordered regions present at its amino-terminus and is independent of protein kinase A. Finally, we demonstrate that nuclear Exchange Protein Activated by cyclic AMP 1 condensates assemble at genomic loci on chromosome 6 in the proximity of Histone Locus Bodies and promote the transcription of a histone gene cluster. Collectively, our data reveal an unexpected mechanism through which cyclic AMP contributes to nuclear spatial compartmentalization and promotes the transcription of specific genes.

(1) Background: Store-operated Ca²⁺ entry (SOCE) drives the cytotoxic activity of cytotoxic T lymphocytes (CTLs) against cancer cells. However, SOCE can be enhanced in cancer cells due to an increase in the expression and/or function of its underlying molecular components, i.e., STIM1 and Orai1. Herein, we evaluated the SOCE expression and function in tumour-infiltrating lymphocytes (TILs) from metastatic colorectal cancer (mCRC) patients. (2) Methods: Functional studies were conducted in TILs expanded ex vivo from CRC liver metastases. Peripheral blood T cells from healthy donors (hPBTs) and mCRC patients (cPBTs) were used as controls. (3) Results: SOCE amplitude is enhanced in TILs compared to hPBTs and cPBTs, but the STIM1 protein is only up-regulated in TILs. Pharmacological manipulation showed that the increase in SOCE mainly depends on tonic modulation by diacylglycerol kinase, which prevents the protein kinase C-dependent inhibition of SOCE activity. The larger SOCE caused a stronger Ca²⁺ response to T-cell receptor stimulation by autologous mCRC cells. Reducing Ca²⁺ influx with BTP-2 during target cell killing significantly increases cytotoxic activity at low target:effector ratios. (4) Conclusions: SOCE is enhanced in ex vivo-expanded TILs deriving from mCRC patients but decreasing Ca²⁺ influx with BTP-2 increases cytotoxic activity at a low TIL density.

The spatiotemporal interplay between the second messenger cyclic AMP (cAMP) and its main effector, protein kinase A (PKA), is crucial for the pleiotropic nature of this cascade. To maintain a high degree of specificity, the cAMP/PKA axis is organised into functional units called microdomains, precisely distributed within the cell. While the subcellular allocation of PKA is guaranteed by a family of tethers called A-Kinase-anchoring Proteins (AKAPs), the mechanisms underlying the efficient confinement of a microdomain’s functional effects are not fully understood. Here, we used FRET-based sensors to detect cAMP levels and PKA-dependent phosphorylation within specific subcellular compartments. We find that cellular cAMP levels may depend on different mechanisms and are responsible for the activation of local PKA enzymes. On the other hand, the dephosphorylating actions of phosphatases dictate the duration of the microdomain’s effects. To test the range of action of PKA microdomains, we used rigid aminoacidic nanorulers to distance our FRET sensors from their original location for 10 or 30 nm. Interestingly, we established that cAMP levels do not affect the spatial range of the microdomain while on the contrary, phosphatase activity provides a functional boundary for phosphorylated PKA targets. Finally, using the same strategy to distance phosphatases from the mitochondria, we found that enzymes close to the outer mitochondrial membrane produced a fragmented phenotype that was not observed when phosphatases were moved to 30 nm from the organelle’s surface. Our findings contribute to the design of a picture where 2 microdomain-forming events have distinct roles. Cyclic AMP elevations trigger the initial activation of subcellular PKA moieties, while the temporal and spatial extent of the PKA’s actions are regulated by phosphatases.

The second messenger 3',5'-cyclic adenosine monophosphate (cAMP) regulates many nuclear processes including transcription1,2, pre-mRNA splicing3 and mitosis4. While most functions are attributed to protein kinase A (PKA)5,6, accumulating evidence suggests that not all nuclear cAMP-dependent effects are mediated by this kinase7, implying that other effectors are involved. Here we explore the nuclear roles of Exchange Protein Activated by cAMP 1 (EPAC1). We find that EPAC1 enters the nucleus through the synergy of two aminoacidic domains and there, in response to cAMP, forms reversible biomolecular condensates through liquid-liquid phase separation. This phenomenon depends on intrinsically disordered regions present at its amino-terminus and is independent of PKA. Finally, we demonstrate that nuclear EPAC1 condensates assemble at genomic loci on chromosome 6 and promote the transcription of a histone gene cluster. Collectively, our data reveal an unexpected mechanism through which cAMP contributes to nuclear spatial compartmentalization and promotes the transcription of specific genes.