Mitochondria Encapsulation in Hydrogel-Based Artificial Cells as ATP Producing Subunits.

Artificial cells (ACs) aim to mimic selected structural and functional features of mammalian cells. In this context, energy generation is an important challenge to be addressed when self-sustained systems are desired. Here, mitochondria isolated from HepG2 cells are employed as natural subunits that facilitate chemically driven adenosine triphosphate (ATP) synthesis. The successful mitochondria isolation is confirmed by monitoring the preserved inner membrane potential, the respiration, and the ATP production ability. The encapsulation of the isolated mitochondria in gelatin-based hydrogels results in similar initial ATP production compared to mitochondria in solution with a sustained ATP production over 24 h. Furthermore, luciferase is coencapsulated with the mitochondria in gelatin-based particles to create ACs and employ the in situ produced ATP to drive the catalytic conversion of d-luciferin. The coencapsulation of luciferase-loaded liposomes with mitochondria in gelatin-based hydrogels is additionally explored where the encapsulation of mitochondria and liposomes resulted in clustering effects that are likely contributing to the functional performance of the active entities. Taken together, mitochondria show potential in cell mimicry to facilitate energy-dependent processes.

Mitochondrial communication in the context of aging.

Mitochondria constantly contribute to the cell homeostasis and this, during the lifespan of a cell, takes its toll. Indeed, the functional decline of mitochondria appears correlated to the aging of the cell. The initial idea was that excessive production of reactive oxygen species (ROS) by functionally compromised mitochondria was the causal link between the decline of the organelle functions and cellular aging. However, in recent years accumulating evidence suggests that the contribution of mitochondria to cellular aging goes beyond ROS production. In this short review, we discuss how intracellular signalling, specifically the cAMP-signalling cascade, is involved in the regulation of mitochondrial functions and potentially in the processes that link mitochondrial status to cellular aging.

Phosphatases control PKA-dependent functional microdomains at the outer mitochondrial membrane.

Evidence supporting the heterogeneity in cAMP and PKA signaling is rapidly accumulating and has been largely attributed to the localization or activity of adenylate cyclases, phosphodiesterases, and A-kinase-anchoring proteins in different cellular subcompartments. However, little attention has been paid to the possibility that, despite homogeneous cAMP levels, a major heterogeneity in cAMP/PKA signaling could be generated by the spatial distribution of the final terminators of this cascade, i.e., the phosphatases. Using FRET-based sensors to monitor cAMP and PKA-dependent phosphorylation in the cytosol and outer mitochondrial membrane (OMM) of primary rat cardiomyocytes, we demonstrate that comparable cAMP increases in these two compartments evoke higher levels of PKA-dependent phosphorylation in the OMM. This difference is most evident for small, physiological increases of cAMP levels and with both OMM-located probes and endogenous OMM proteins. We demonstrate that this disparity depends on differences in the rates of phosphatase-dependent dephosphorylation of PKA targets in the two compartments. Furthermore, we show that the activity of soluble phosphatases attenuates PKA-driven activation of the cAMP response element-binding protein while concurrently enhancing PKA-dependent mitochondrial elongation. We conclude that phosphatases can sculpt functionally distinct cAMP/PKA domains even in the absence of gradients or microdomains of this messenger. We present a model that accounts for these unexpected results in which the degree of PKA-dependent phosphorylation is dictated by both the subcellular distribution of the phosphatases and the different accessibility of membrane-bound and soluble phosphorylated substrates to the cytosolic enzymes.

Shaping mitochondrial dynamics: The role of cAMP signalling.

In recent years, our idea of mitochondria evolved from "mere" energy and metabolite producers to key regulators of many cellular functions. In order to preserve and protect their functional status, these organelles engage a number of dynamic processes that allow them to decrease accumulated burden and maintain their homeostasis. Indeed, mitochondria can unite (fusion), divide (fission), position themselves strategically in the cell (motility/trafficking) and if irreversibly damaged or dysfunctional eliminated (mitophagy). These dynamic processes can be controlled both by mitochondrial and cellular signalling pathways, hence allowing mitochondria to tune their function to the cellular needs. Among the regulatory mechanisms, reversible phosphorylation downstream the cyclic AMP (cAMP) signalling cascade was shown to deeply influence mitochondrial dynamics. This review explores the emerging evidence suggesting that cAMP is a key player in the orchestration of mitochondrial fusion/fission, motility and mitophagy, extending the repertoire of this second messenger, which is now recognised as a major regulator of mitochondrial homeostasis.

Dysregulation of Neuronal Ca2+ Channel Linked to Heightened Sympathetic Phenotype in Prohypertensive States.

UNLABELLED: Hypertension is associated with impaired nitric oxide (NO)-cyclic nucleotide (CN)-coupled intracellular calcium (Ca(2+)) homeostasis that enhances cardiac sympathetic neurotransmission. Because neuronal membrane Ca(2+) currents are reduced by NO-activated S-nitrosylation, we tested whether CNs affect membrane channel conductance directly in neurons isolated from the stellate ganglia of spontaneously hypertensive rats (SHRs) and their normotensive controls. Using voltage-clamp and cAMP-protein kinase A (PKA) FRET sensors, we hypothesized that impaired CN regulation provides a direct link to abnormal signaling of neuronal calcium channels in the SHR and that targeting cGMP can restore the channel phenotype. We found significantly larger whole-cell Ca(2+) currents from diseased neurons that were largely mediated by the N-type Ca(2+) channel (Cav2.2). Elevating cGMP restored the SHR Ca(2+) current to levels seen in normal neurons that were not affected by cGMP. cGMP also decreased cAMP levels and PKA activity in diseased neurons. In contrast, cAMP-PKA activity was increased in normal neurons, suggesting differential switching in phosphodiesterase (PDE) activity. PDE2A inhibition enhanced the Ca(2+) current in normal neurons to a conductance similar to that seen in SHR neurons, whereas the inhibitor slightly decreased the current in diseased neurons. Pharmacological evidence supported a switching from cGMP acting via PDE3 in control neurons to PDE2A in SHR neurons in the modulation of the Ca(2+) current. Our data suggest that a disturbance in the regulation of PDE-coupled CNs linked to N-type Ca(2+) channels is an early hallmark of the prohypertensive phenotype associated with intracellular Ca(2+) impairment underpinning sympathetic dysautonomia. SIGNIFICANCE STATEMENT: Here, we identify dysregulation of cyclic-nucleotide (CN)-linked neuronal Ca(2+) channel activity that could provide the trigger for the enhanced sympathetic neurotransmission observed in the prohypertensive state. Furthermore, we provide evidence that increasing cGMP rescues the channel phenotype and restores ion channel activity to levels seen in normal neurons. We also observed CN cross-talk in sympathetic neurons that may be related to a differential switching in phosphodiesterase activity. The presence of these early molecular changes in asymptomatic, prohypertensive animals could facilitate the identification of novel therapeutic targets with which to modulate intracellular Ca(2+) Turning down the gain of sympathetic hyperresponsiveness in cardiovascular disease associated with sympathetic dysautonomia would have significant therapeutic utility.

Cardiac Hypertrophy Is Inhibited by a Local Pool of cAMP Regulated by Phosphodiesterase 2.

RATIONALE: Chronic elevation of 3'-5'-cyclic adenosine monophosphate (cAMP) levels has been associated with cardiac remodeling and cardiac hypertrophy. However, enhancement of particular aspects of cAMP/protein kinase A signaling seems to be beneficial for the failing heart. cAMP is a pleiotropic second messenger with the ability to generate multiple functional outcomes in response to different extracellular stimuli with strict fidelity, a feature that relies on the spatial segregation of the cAMP pathway components in signaling microdomains. OBJECTIVE: How individual cAMP microdomains affect cardiac pathophysiology remains largely to be established. The cAMP-degrading enzymes phosphodiesterases (PDEs) play a key role in shaping local changes in cAMP. Here we investigated the effect of specific inhibition of selected PDEs on cardiac myocyte hypertrophic growth. METHODS AND RESULTS: Using pharmacological and genetic manipulation of PDE activity, we found that the rise in cAMP resulting from inhibition of PDE3 and PDE4 induces hypertrophy, whereas increasing cAMP levels via PDE2 inhibition is antihypertrophic. By real-time imaging of cAMP levels in intact myocytes and selective displacement of protein kinase A isoforms, we demonstrate that the antihypertrophic effect of PDE2 inhibition involves the generation of a local pool of cAMP and activation of a protein kinase A type II subset, leading to phosphorylation of the nuclear factor of activated T cells. CONCLUSIONS: Different cAMP pools have opposing effects on cardiac myocyte cell size. PDE2 emerges as a novel key regulator of cardiac hypertrophy in vitro and in vivo, and its inhibition may have therapeutic applications.

Beyond Intracellular Signaling: The Ins and Outs of Second Messengers Microdomains.

A typical characteristic of eukaryotic cells compared to prokaryotes is represented by the spatial heterogeneity of the different structural and functional components: for example, most of the genetic material is surrounded by a highly specific membrane structure (the nuclear membrane), continuous with, yet largely different from, the endoplasmic reticulum (ER); oxidative phosphorylation is carried out by organelles enclosed by a double membrane, the mitochondria; in addition, distinct domains, enriched in specific proteins, are present in the plasma membrane (PM) of most cells. Less obvious, but now generally accepted, is the notion that even the concentration of small molecules such as second messengers (Ca2+ and cAMP in particular) can be highly heterogeneous within cells. In the case of most organelles, the differences in the luminal levels of second messengers depend either on the existence on their membrane of proteins that allow the accumulation/release of the second messenger (e.g., in the case of Ca2+, pumps, exchangers or channels), or on the synthesis and degradation of the specific molecule within the lumen (the autonomous intramitochondrial cAMP system). It needs stressing that the existence of a surrounding membrane does not necessarily imply the existence of a gradient between the cytosol and the organelle lumen. For example, the nuclear membrane is highly permeable to both Ca2+ and cAMP (nuclear pores are permeable to solutes up to 50 kDa) and differences in [Ca2+] or [cAMP] between cytoplasm and nucleoplasm are not seen in steady state and only very transiently during cell activation. A similar situation has been observed, as far as Ca2+ is concerned, in peroxisomes.

Protein kinase inhibitor ß enhances the constitutive activity of G-protein-coupled zinc receptor GPR39.

GPR39 is a G-protein-coupled zinc receptor that protects against diverse effectors of cell death. Its protective activity is mediated via constitutive activation of Gα13 and the RhoA pathway, leading to increased SRE (serum-response element)-dependent transcription; the zinc-dependent immediate activation of GPR39 involves Gq-mediated increases in cytosolic Ca2+ and Gs coupling leading to increased cAMP levels. We used the cytosolic and soluble C-terminus of GPR39 in a Y2H (yeast-2-hybrid) screen for interacting proteins, thus identifying PKIB (protein kinase A inhibitor ß). Co-expression of GPR39 with PKIB increased the protective activity of GPR39 via the constitutive, but not the ligand-mediated, pathway. PKIB inhibits protein kinase A by direct interaction with its pseudosubstrate domain; mutation of this domain abolished the inhibitory activity of PKIB on protein kinase A activity, but had no effect on the interaction with GPR39, cell protection and induction of SRE-dependent transcription. Zinc caused dissociation of PKIB from GPR39, thereby liberating it to associate with protein kinase A and inhibit its activity, which would result in a negative-feedback loop with the ability to limit activation of the Gs pathway by zinc.

cAMP signalling meets mitochondrial compartments.

Mitochondria are highly dynamic organelles comprising at least three distinct areas, the OMM (outer mitochondrial membrane), the IMS (intermembrane space) and the mitochondrial matrix. Physical compartmentalization allows these organelles to host different functional domains and therefore participate in a variety of important cellular actions such as ATP synthesis and programmed cell death. In a surprising homology, it is now widely accepted that the ubiquitous second messenger cAMP uses the same stratagem, compartmentalization, in order to achieve the characteristic functional pleiotropy of its pathway. Accumulating evidence suggests that all the main mitochondrial compartments contain segregated cAMP cascades; however, the regulatory properties and functional significance of such domains are not fully understood and often remain controversial issues. The present mini-review discusses our current knowledge of how the marriage between mitochondrial and cAMP compartmentalization is achieved and its effects on the biology of the cell.

Cyclic AMP induces reversible EPAC1 condensates that regulate histone transcription.

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.

Organic Selenium induces ferroptosis in pancreatic cancer cells.

Pancreatic ductal adenocarcinoma (PDA) cells reprogram both mitochondrial and lysosomal functions to support growth. At the same time, this causes significant dishomeostasis of free radicals. While this is compensated by the upregulation of detoxification mechanisms, it also represents a potential vulnerability. Here we demonstrate that PDA cells are sensitive to the inhibition of the mevalonate pathway (MVP), which supports the biosynthesis of critical antioxidant intermediates and protect from ferroptosis. We attacked the susceptibility of PDA cells to ferroptotic death with selenorganic compounds, including dibenzyl diselenide (DBDS) that exhibits potent pro-oxidant properties and inhibits tumor growth in vitro and in vivo. DBDS treatment induces the mobilization of iron from mitochondria enabling uncontrolled lipid peroxidation. Finally, we showed that DBDS and statins act synergistically to promote ferroptosis and provide evidence that combined treatment is a viable strategy to combat PDA.

Termination and activation of store-operated cyclic AMP production.

Diverse pathophysiological processes (e.g. obesity, lifespan determination, addiction and male fertility) have been linked to the expression of specific isoforms of the adenylyl cyclases (AC1-AC10), the enzymes that generate cyclic AMP (cAMP). Our laboratory recently discovered a new mode of cAMP production, prominent in certain cell types, that is stimulated by any manoeuvre causing reduction of free [Ca(2+) ] within the lumen of the endoplasmic reticulum (ER) calcium store. Activation of this 'store-operated' pathway requires the ER Ca(2+) sensor, STIM1, but the identity of the enzymes responsible for cAMP production and how this process is regulated is unknown. Here, we used sensitive FRET-based sensors for cAMP in single cells combined with silencing and overexpression approaches to show that store-operated cAMP production occurred preferentially via the isoform AC3 in NCM460 colonic epithelial cells. Ca(2+) entry via the plasma membrane Ca(2+) channel, Orai1, suppressed cAMP production, independent of store refilling. These findings are an important first step towards defining the functional significance and to identify the protein composition of this novel Ca(2+) /cAMP crosstalk system.

The sumoylation pathway is dysregulated in multiple myeloma and is associated with adverse patient outcome.

Multiple myeloma (MM) is a plasma cell neoplasm that proceeds through a premalignant state of monoclonal gammopathy of unknown significance; however, the molecular events responsible for myelomagenesis remain uncharacterized. To identify cellular pathways deregulated in MM, we addressed that sumoylation is homologous to ubiquitination and results in the attachment of the ubiquitin-like protein Sumo onto target proteins. Sumoylation was markedly enhanced in MM patient lysates compared with normal plasma cells and expression profiling indicated a relative induction of sumoylation pathway genes. The Sumo-conjugating enzyme Ube2I, the Sumo-ligase PIAS1, and the Sumo-inducer ARF were elevated in MM patient samples and cell lines. Survival correlated with expression because 80% of patients with low UBE2I and PIAS1 were living 6 years after transplantation, whereas only 45% of patients with high expression survived 6 years. UBE2I encodes the sole Sumo-conjugating enzyme in mammalian cells and cells transfected with a dominant-negative sumoylation-deficient UBE2I mutant exhibited decreased survival after radiation exposure, impaired adhesion to bone marrow stroma cell and decreased bone marrow stroma cell-induced proliferation. UBE2I confers cells with multiple advantages to promote tumorigenesis and predicts decreased survival when combined with PIAS1. The sumoylation pathway is a novel therapeutic target with implications for existing proteasomal-based treatment strategies.

The Readthrough Isoform AQP4ex Is Constitutively Phosphorylated in the Perivascular Astrocyte Endfeet of Human Brain.

AQP4ex is a recently discovered isoform of AQP4 generated by a translational readthrough mechanism. It is strongly expressed at the astrocyte perivascular endfeet as a component of the supramolecular membrane complex, commonly called orthogonal array of particles (OAP), together with the canonical isoforms M1 and M23 of AQP4. Previous site-directed mutagenesis experiments suggested the potential role of serine331 and serine335, located in the extended peptide of AQP4ex, in water channel activity by phosphorylation. In the present study we evaluated the effective phosphorylation of human AQP4ex. A small scale bioinformatic analysis indicated that only Ser335 is conserved in human, mouse and rat AQP4ex. The phosphorylation site of Ser335 was assessed through generation of phospho-specific antibodies in rabbits. Antibody specificity was first evaluated in binding phosphorylated peptide versus its unphosphorylated analog by ELISA, which was further confirmed by site-directed mutagenesis experiments. Western blot and immunofluorescence experiments revealed strong expression of phosphorylated AQP4ex (p-AQP4ex) in human brain and localization at the perivascular astrocyte endfeet in supramolecular assemblies identified by BN/PAGE experiments. All together, these data reveal, for the first time, the existence of a phosphorylated form of AQP4, at Ser335 in the extended sequence exclusive of AQP4ex. Therefore, we anticipate an important physiological role of p-AQP4ex in human brain water homeostasis.

Abnormal Cyclic Nucleotide Signaling at the Outer Mitochondrial Membrane In Sympathetic Neurons During the Early Stages of Hypertension.

BACKGROUND: Disruption of cyclic nucleotide signaling in sympathetic postganglionic neurons contributes to impaired intracellular calcium handling (Ca2+) and the development of dysautonomia during the early stages of hypertension, although how this occurs is poorly understood. Emerging evidence supports the uncoupling of signalosomes in distinct cellular compartments involving cyclic nucleotide-sensitive PDEs (phosphodiesterases), which may underpin the autonomic phenotype in stellate neurons. METHODS: Using a combination of single-cell RNA sequencing together with Forster resonance energy transfer-based sensors to monitor cyclic adenosine 3',5'-monophosphate, PKA (protein kinase A)-dependent phosphorylation and cGMP (cyclic guanosine 3',5'-monophosphate), we tested the hypothesis that dysregulation occurs in a sub-family of PDEs in the cytosol and outer mitochondrial membrane of neurons from the stellate ganglion. RESULTS: PDE2A, 6D, 7A, 9A genes were highly expressed in young Wistar neurons and also conserved in neurons from spontaneously hypertensive rats (SHRs). In stellate neurons from prehypertensive SHRs, we found the levels of cyclic adenosine 3',5'-monophosphate and cGMP at the outer mitochondrial membrane were decreased compared with normal neurons. The reduced cyclic adenosine 3',5'-monophosphate response was due to the hydrolytic activity of overexpressed PDE2A2 located at the mitochondria. Normal cyclic adenosine 3',5'-monophosphate levels were re-established by inhibition of PDE2A. There was also a greater PKA-dependent phosphorylation in the cytosol and at the outer mitochondrial membrane in spontaneously hypertensive rat neurons, where this response was regulated by protein phosphatases. The cGMP response was only restored by inhibition of PDE6. CONCLUSIONS: When taken together, these results suggest that site-specific inhibition of PDE2A and PDE6D at the outer mitochondrial membrane may provide a therapeutic target to ameliorate cardiac sympathetic impairment during the onset of hypertension.

Store-Operated Ca2+ Entry Is Up-Regulated in Tumour-Infiltrating Lymphocytes from Metastatic Colorectal Cancer Patients.

(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 GIP/GIPR axis in medullary thyroid cancer: clinical and molecular findings.

The improper expression of glucose-dependent insulinotropic polypeptide receptor (GIPR) and the GIP/GIPR axis activation has been increasingly recognized in endocrine tumors, with a potential diagnostic and prognostic value. A high tumor-to-normal tissue ratio (T/N ratio) of GIPR was reported both in humans' and in rats' medullary thyroid cancer (MTC), suggesting a direct link between the neoplastic transformation and the mechanism of receptor overexpression. In this study, we evaluated the potential diagnostic and prognostic significance of GIPR expression in a large cohort of MTC patients by correlating GIPR mRNA steady-state levels to clinical phenotypes. The molecular effect of GIP/GIPR axis stimulation in MTC-derived cells was also determined. We detected GIPR expression in ~80% of tumor specimens, especially in sporadic, larger, advanced-stage cancers with higher Ki-67 values. GIPR stimulation induced cAMP elevation in MTC-derived cells and a small but significant fluctuation in Ca2+, both likely associated with increased calcitonin secretion. On the contrary, the effects on PI3K-Akt and MAPK-ERK1/2 signaling pathways were marginal. To conclude, our data confirm the high T/N GIPR ratio in MTC tumors and suggest that it may represent an index for the degree of advancement of the malignant process. We have also observed a functional coupling between GIP/GIPR axis and calcitonin secretion in MTC models. However, the molecular mechanisms underlying this process and the possible implication of GIP/GIPR axis activation in MTC diagnosis and prognosis need further evaluation.

PRKA/PKA signals and autophagy: space matters.

Macroautophagy/autophagy is the cellular process responsible for the elimination and recycling of aggregated proteins and damaged organelles. Whereas autophagy is strictly regulated by several signaling cascades, the link between this process and the subcellular distribution of its regulatory pathways remains to be established. Our recent work suggests that the compartmentalization of PRKA/PKA (protein kinase cAMP-activated) determines its effects on autophagy. We found that increased cAMP levels generate dramatically different PRKA activity "signatures" mainly dependent on the actions of phosphatases and the distribution of the PRKA holoenzymes containing type II regulatory subunits (PRKAR2A and PRKAR2B; RII). In this punctum we discuss how compartmentalized PRKA signaling events are generated and affect the autophagic flux in specific cell types.

The basics of mitochondrial cAMP signalling: Where, when, why.

Cytosolic cAMP signalling in live cells has been extensively investigated in the past, while only in the last decade the existence of an intramitochondrial autonomous cAMP homeostatic system began to emerge. Thanks to the development of novel tools to investigate cAMP dynamics and cAMP/PKA-dependent phosphorylation within the matrix and in other mitochondrial compartments, it is now possible to address directly and in intact living cells a series of questions that until now could be addressed only by indirect approaches, in isolated organelles or through subcellular fractionation studies. In this contribution we discuss the mechanisms that regulate cAMP dynamics at the surface and inside mitochondria, and its crosstalk with organelle Ca2+ handling. We then address a series of still unsolved questions, such as the intramitochondrial localization of key elements of the cAMP signaling toolkit, e.g., adenylate cyclases, phosphodiesterases, protein kinase A (PKA) and Epac. Finally, we discuss the evidence for and against the existence of an intramitochondrial PKA pool and the functional role of cAMP increases within the organelle matrix.

PKA compartmentalization links cAMP signaling and autophagy.

Autophagy is a highly regulated degradative process crucial for maintaining cell homeostasis. This important catabolic mechanism can be nonspecific, but usually occurs with fine spatial selectivity (compartmentalization), engaging only specific subcellular sites. While the molecular machines driving autophagy are well understood, the involvement of localized signaling events in this process is not well defined. Among the pathways that regulate autophagy, the cyclic AMP (cAMP)/protein kinase A (PKA) cascade can be compartmentalized in distinct functional units called microdomains. However, while it is well established that, depending on the cell type, cAMP can inhibit or promote autophagy, the role of cAMP/PKA microdomains has not been tested. Here we show not only that the effects on autophagy of the same cAMP elevation differ in different cell types, but that they depend on a highly complex sub-compartmentalization of the signaling cascade. We show in addition that, in HT-29 cells, in which autophagy is modulated by cAMP rising treatments, PKA activity is strictly regulated in space and time by phosphatases, which largely prevent the phosphorylation of soluble substrates, while membrane-bound targets are less sensitive to the action of these enzymes. Interestingly, we also found that the subcellular distribution of PKA type-II regulatory PKA subunits hinders the effect of PKA on autophagy, while displacement of type-I regulatory PKA subunits has no effect. Our data demonstrate that local PKA activity can occur independently of local cAMP concentrations and provide strong evidence for a link between localized PKA signaling events and autophagy.