cAMP signaling: a remarkably regional affair.

Louis Pasteur once famously said 'in the fields of observation chance favors only the prepared mind'. Much of chance is being in the right place at the right time. This is particularly true in the crowded molecular environment of the cell where being in the right place is often more important than timing. Although Brownian motion argues that enzymes will eventually bump into substrates, this probability is greatly enhanced if both molecules reside in the same subcellular compartment. However, activation of cell signaling enzymes often requires the transmission of chemical signals from extracellular stimuli to intracellular sites of action. This review highlights new developments in our understanding of cAMP generation and the 3D utilization of this second messenger inside cells.

Signalling scaffolds and local organization of cellular behaviour.

Cellular responses to environmental cues involve the mobilization of GTPases, protein kinases and phosphoprotein phosphatases. The spatial organization of these signalling enzymes by scaffold proteins helps to guide the flow of molecular information. Allosteric modulation of scaffolded enzymes can alter their catalytic activity or sensitivity to second messengers in a manner that augments, insulates or terminates local cellular events. This Review examines the features of scaffold proteins and highlights examples of locally organized groups of signalling enzymes that drive essential physiological processes, including hormone action, heart rate, cell division, organelle movement and synaptic transmission.

Anchored PKA synchronizes adrenergic phosphoregulation of cardiac Cav1.2 channels.

Adrenergic modulation of voltage gated Ca2+ currents is a context specific process. In the heart Cav1.2 channels initiate excitation-contraction coupling. This requires protein kinase A (PKA) phosphorylation of the small GTPase Rad (Ras associated with diabetes) and involves direct phosphorylation of a1 subunit of the Cav1.2 at Ser1700. A contributing factor is the proximity of PKA to the channel through association with A-kinase anchoring proteins (AKAPs). Disruption of PKA anchoring by the disruptor peptide AKAP-IS prevents up-regulation of Cav1.2 currents in tsA-201 cells. Biochemical analyses demonstrate that Rad does not function as an A-kinase anchoring protein. Electrophysiological recording shows that channel mutants lacking phosphorylation sites (Cav1.2 STAA) lose responsivity to the second messenger cAMP. Measurements in cardiomyocytes isolated from Rad-/- mice show that adrenergic activation of Cav1.2 is attenuated but not completely abolished. Whole animal electrocardiography studies reveal that cardiac selective Rad knockout mice exhibited higher baseline left-ventricular ejection fraction (EF), greater fractional shortening (FS), and increased heart rate as compared to control animals. Yet, each parameter of cardiac function was slightly elevated when Rad-/- mice were treated with the adrenergic agonist isoproterenol. Thus, phosphorylation of Cav1.2 and dissociation of phospho-Rad from the channel are local cAMP responsive events that act in concert to enhance L-type calcium currents. This convergence of local PKA regulatory events at the cardiac L-type calcium channel may permit maximal ß-adrenergic influence on the fight-or-flight response.

Drugs That Regulate Local Cell Signaling: AKAP Targeting as a Therapeutic Option.

Cells respond to environmental cues by mobilizing signal transduction cascades that engage protein kinases and phosphoprotein phosphatases. Correct organization of these enzymes in space and time enables the efficient and precise transmission of chemical signals. The cyclic AMP-dependent protein kinase A is compartmentalized through its association with A-kinase anchoring proteins (AKAPs). AKAPs are a family of multivalent scaffolds that constrain signaling enzymes and effectors at subcellular locations to drive essential physiological events. More recently, it has been recognized that defective signaling in certain endocrine disorders and cancers proceeds through pathological AKAP complexes. Consequently, pharmacologically targeting these macromolecular complexes unlocks new therapeutic opportunities for a growing number of clinical indications. This review highlights recent findings on AKAP signaling in disease, particularly in certain cancers, and offers an overview of peptides and small molecules that locally regulate AKAP-binding partners.

Classification of Cushing's syndrome PKAc mutants based upon their ability to bind PKI.

Cushing's syndrome is an endocrine disorder caused by excess production of the stress hormone cortisol. Precision medicine strategies have identified single allele mutations within the PRKACA gene that drive adrenal Cushing's syndrome. These mutations promote perturbations in the catalytic core of protein kinase A (PKAc) that impair autoinhibition by regulatory subunits and compartmentalization via recruitment into AKAP signaling islands. PKAcL205R is found in ∼45% of patients, whereas PKAcE31V, PKAcW196R, and L198insW and C199insV insertion mutants are less prevalent. Mass spectrometry, cellular, and biochemical data indicate that Cushing's PKAc variants fall into two categories: those that interact with the heat-stable protein kinase inhibitor PKI, and those that do not. In vitro activity measurements show that wild-type PKAc and W196R activities are strongly inhibited by PKI (IC50 < 1 nM). In contrast, PKAcL205R activity is not blocked by the inhibitor. Immunofluorescent analyses show that the PKI-binding variants wild-type PKAc, E31V, and W196R are excluded from the nucleus and protected against proteolytic processing. Thermal stability measurements reveal that upon co-incubation with PKI and metal-bound nucleotide, the W196R variant tolerates melting temperatures 10°C higher than PKAcL205. Structural modeling maps PKI-interfering mutations to a ∼20 Šdiameter area at the active site of the catalytic domain that interfaces with the pseudosubstrate of PKI. Thus, Cushing's kinases are individually controlled, compartmentalized, and processed through their differential association with PKI.

Phosphorylation, compartmentalization, and cardiac function.

Protein phosphorylation is a fundamental element of cell signaling. First discovered as a biochemical switch in glycogen metabolism, we now know that this posttranslational modification permeates all aspects of cellular behavior. In humans, over 540 protein kinases attach phosphate to acceptor amino acids, whereas around 160 phosphoprotein phosphatases remove phosphate to terminate signaling. Aberrant phosphorylation underlies disease, and kinase inhibitor drugs are increasingly used clinically as targeted therapies. Specificity in protein phosphorylation is achieved in part because kinases and phosphatases are spatially organized inside cells. A prototypic example is compartmentalization of the cyclic adenosine 3',5'-monophosphate (cAMP)-dependent protein kinase A through association with A-kinase anchoring proteins. This configuration creates autonomous signaling islands where the anchored kinase is constrained in proximity to activators, effectors, and selected substates. This article primarily focuses on A kinase anchoring protein (AKAP) signaling in the heart with an emphasis on anchoring proteins that spatiotemporally coordinate excitation-contraction coupling and hypertrophic responses.

Training the healthcare workforce: the global experience with telementorship for hepatitis B and hepatitis C.

BACKGROUND: Telementorship has emerged as an innovative strategy to decentralise medical knowledge and increase healthcare capacity across a wide range of disease processes. We report the global experience with telementorship to support healthcare workers delivering hepatitis B virus (HBV) and hepatitis C virus (HCV) care and treatment. METHODS: In early 2020, we conducted a survey of HBV and HCV telementorship programmes, followed by an in-depth interview with programme leads. Programmes were eligible to participate if they were located outside of the United States (U.S.), focused on support to healthcare workers in management of HBV and/or HCV, and were affiliated with or maintained adherence to the Project ECHO model, a telementorship programme pioneered at the University of New Mexico. One programme in the U.S., focused on HCV treatment in the Native American community, was purposively sampled and invited to participate. Surveys were administered online, and all qualitative interviews were performed remotely. Descriptive statistics were calculated for survey responses, and qualitative interviews were assessed for major themes. RESULTS: Eleven of 18 eligible programmes completed the survey and follow up interview. Sixty-four percent of programmes were located at regional academic medical centers. The majority of programmes (64%) were led by hepatologists. Most programmes (82%) addressed both HBV and HCV, and the remainder focused on HCV only. The median number of participating clinical spoke sites per programme was 22, and most spoke site participants were primary care providers. Most ECHO sessions were held monthly (36%) or bimonthly (27%), with sessions ranging from 45 min to 2 h in length. Programme leaders identified collective learning, empowerment and collaboration to be key strengths of their telementorship programme, while insufficient funding and a lack of protected time for telementorship leaders and participants were identified as major barriers to success. CONCLUSION: The Project ECHO model for telementorship can be successfully implemented across high and low-and-middle-income countries to improve provider knowledge and experience in management of viral hepatitis. There is a tremendous opportunity to further expand upon the existing experience with telementorship to support non-specialist healthcare workers and promote elimination of viral hepatitis.

Training the healthcare workforce to support task-shifting and viral hepatitis elimination: a global review of English language online trainings and in-person workshops for management of hepatitis B and C infection.

BACKGROUND: Achieving World Health Organization (WHO) targets for viral hepatitis elimination will require simplification and decentralisation of care, supported through task-shifting and training of non-specialist frontline healthcare workers. To inform development of national health worker trainings in viral hepatitis, we review and summarise available online and workshop trainings for management of hepatitis B virus (HBV) and hepatitis C virus (HCV). METHODS: We performed a systematic search of PubMed, Embase, Web of Science, conference abstracts, and grey literature using Google to identify online and in-person workshop trainings for health workers focused on HBV and/or HCV. Additional trainings were identified through a WHO regional network. We included online trainings written in English and in-person workshops developed for low-and-middle-income countries (LMICs). Available curricula are summarised together with key operational features (e.g. training length, year developed/updated, developing institution) and programmatic features (e.g. content, mechanism for self-assessment, use of clinical case studies). RESULTS: A total of 30 trainings met our inclusion criteria (10 online trainings; 20 in-person workshops). 50% covered both HBV and HCV, 13% HBV alone and 37% HCV alone. Among online trainings, only 2 (20%) were specifically developed or adapted for LMICs; 70% covered all aspects of hepatitis care, including prevention, assessment, and treatment; 9 (90%) included guidance on when to refer to specialists, and 6 (60%) included modules on management in specific populations (e.g., people who inject drugs [PWID], prisoners, and children). Online trainings used different formats including text-based modules, narrated slide-sets, and interactive web-based modules. Most workshops (95%) were targeted towards non-specialty providers, and 50% were an integral part of a national strategy for viral hepatitis elimination. Workshop length ranged from several hours to multiple sessions over the course of months, and many were part of a blended educational model, which included other opportunities for ongoing learning (e.g., telementorship). CONCLUSION: This compendium of online and in-person workshop trainings for HBV and HCV is a useful resource for national hepatitis programmes developing training curricula for non-specialists. Additional online training curricula are needed for use in LMICs, and additional materials are needed to address management challenges in key populations, such as PWID.

Beyond PKA: Evolutionary and structural insights that define a docking and dimerization domain superfamily.

Protein-interaction domains can create unique macromolecular complexes that drive evolutionary innovation. By combining bioinformatic and phylogenetic analyses with structural approaches, we have discovered that the docking and dimerization (D/D) domain of the PKA regulatory subunit is an ancient and conserved protein fold. An archetypal function of this module is to interact with A-kinase-anchoring proteins (AKAPs) that facilitate compartmentalization of this key cell-signaling enzyme. Homology searching reveals that D/D domain proteins comprise a superfamily with 18 members that function in a variety of molecular and cellular contexts. Further in silico analyses indicate that D/D domains segregate into subgroups on the basis of their similarity to type I or type II PKA regulatory subunits. The sperm autoantigenic protein 17 (SPA17) is a prototype of the type II or R2D2 subgroup that is conserved across metazoan phyla. We determined the crystal structure of an extended D/D domain from SPA17 (amino acids 1-75) at 1.72 Å resolution. This revealed a four-helix bundle-like configuration featuring terminal ß-strands that can mediate higher order oligomerization. In solution, SPA17 forms both homodimers and tetramers and displays a weak affinity for AKAP18. Quantitative approaches reveal that AKAP18 binding occurs at nanomolar affinity when SPA17 heterodimerizes with the ropporin-1-like D/D protein. These findings expand the role of the D/D fold as a versatile protein-interaction element that maintains the integrity of macromolecular architectures within organelles such as motile cilia.

Kinase-anchoring proteins in ciliary signal transduction.

Historically, the diffusion of chemical signals through the cell was thought to occur within a cytoplasmic soup bounded by the plasma membrane. This theory was predicated on the notion that all regulatory enzymes are soluble and moved with a Brownian motion. Although enzyme compartmentalization was initially rebuffed by biochemists as a 'last refuge of a scoundrel', signal relay through macromolecular complexes is now accepted as a fundamental tenet of the burgeoning field of spatial biology. A-Kinase anchoring proteins (AKAPs) are prototypic enzyme-organizing elements that position clusters of regulatory proteins at defined subcellular locations. In parallel, the primary cilium has gained recognition as a subcellular mechanosensory organelle that amplifies second messenger signals pertaining to metazoan development. This article highlights advances in our understanding of AKAP signaling within the primary cilium and how defective ciliary function contributes to an increasing number of diseases known as ciliopathies.

Co-ordinated control of the Aurora B abscission checkpoint by PKCε complex assembly, midbody recruitment and retention.

A requirement for PKCε in exiting from the Aurora B dependent abscission checkpoint is associated with events at the midbody, however, the recruitment, retention and action of PKCε in this compartment are poorly understood. Here, the prerequisite for 14-3-3 complex assembly in this pathway is directly linked to the phosphorylation of Aurora B S227 at the midbody. However, while essential for PKCε control of Aurora B, 14-3-3 association is shown to be unnecessary for the activity-dependent enrichment of PKCε at the midbody. This localisation is demonstrated to be an autonomous property of the inactive PKCε D532N mutant, consistent with activity-dependent dissociation. The C1A and C1B domains are necessary for this localisation, while the C2 domain and inter-C1 domain (IC1D) are necessary for retention at the midbody. Furthermore, it is shown that while the IC1D mutant retains 14-3-3 complex proficiency, it does not support Aurora B phosphorylation, nor rescues division failure observed with knockdown of endogenous PKCε. It is concluded that the concerted action of multiple independent events facilitates PKCε phosphorylation of Aurora B at the midbody to control exit from the abscission checkpoint.

Awareness and Correlates of HIV Pre-Exposure Prophylaxis (PrEP) Among HIV-negative People Who Access Syringe Services in Seattle, Washington.

BACKGROUND: HIV pre-exposure prophylaxis (PrEP) is safe and effective for use in people who inject drugs (PWID), but PrEP is underutilized in this population. We assessed awareness of PrEP and correlates of interest in PrEP among PWID in Seattle, Washington. METHODS: This study analyzed data from a 2019 survey of PWID at 3 Seattle-area syringe service programs (SSPs). We used descriptive statistics to compare PrEP-aware and unaware PWID and multivariable Poisson regression with robust standard errors to estimate adjusted prevalence ratios (APR) for interest in PrEP. RESULTS: Among 348 HIV-negative PWID, ≤1% were currently taking PrEP, 51% were PrEP aware and 46% were interested in PrEP. Interest in PrEP was inversely associated with prior PrEP awareness (APR 0.58, 95% CI 0.45 - 0.74); however, interest in PrEP was high among PWID meeting pre-specified risk criteria for HIV (APR 1.41, 95% CI 1.06 - 1.88). CONCLUSIONS: Our results suggest increasing awareness of PrEP may not be sufficient to promote PrEP uptake among PWID, and further efforts are needed to understand perceptions of risk for HIV, determinants of PrEP use, and to investigate successful strategies for PrEP implementation and delivery in this marginalized population.Supplemental data for this article is available online at https://doi.org/10.1080/10826084.2021.2012688 .

Gravin-associated kinase signaling networks coordinate γ-tubulin organization at mitotic spindle poles.

Mitogenic signals that regulate cell division often proceed through multienzyme assemblies within defined intracellular compartments. The anchoring protein Gravin restricts the action of mitotic kinases and cell-cycle effectors to defined mitotic structures. In this report we discover that genetic deletion of Gravin disrupts proper accumulation and asymmetric distribution of γ-tubulin during mitosis. We utilize a new precision pharmacology tool, Local Kinase Inhibition, to inhibit the Gravin binding partner polo-like kinase 1 at spindle poles. Using a combination of gene-editing approaches, quantitative imaging, and biochemical assays, we provide evidence that disruption of local polo-like kinase 1 signaling underlies the γ-tubulin distribution defects observed with Gravin loss. Our study uncovers a new role for Gravin in coordinating γ-tubulin recruitment during mitosis and illuminates the mechanism by which signaling enzymes regulate this process at a distinct subcellular location.

A-kinase-anchoring protein 1 (dAKAP1)-based signaling complexes coordinate local protein synthesis at the mitochondrial surface.

Compartmentalization of macromolecules is a ubiquitous molecular mechanism that drives numerous cellular functions. The appropriate organization of enzymes in space and time enables the precise transmission and integration of intracellular signals. Molecular scaffolds constrain signaling enzymes to influence the regional modulation of these physiological processes. Mitochondrial targeting of protein kinases and protein phosphatases provides a means to locally control the phosphorylation status and action of proteins on the surface of this organelle. Dual-specificity protein kinase A anchoring protein 1 (dAKAP1) is a multivalent binding protein that targets protein kinase A (PKA), RNAs, and other signaling enzymes to the outer mitochondrial membrane. Many AKAPs recruit a diverse set of binding partners that coordinate a broad range of cellular processes. Here, results of MS and biochemical analyses reveal that dAKAP1 anchors additional components, including the ribonucleoprotein granule components La-related protein 4 (LARP4) and polyadenylate-binding protein 1 (PABPC1). Local translation of mRNAs at organelles is a means to spatially control the synthesis of proteins. RNA-Seq data demonstrate that dAKAP1 binds mRNAs encoding proteins required for mitochondrial metabolism, including succinate dehydrogenase. Functional studies suggest that the loss of dAKAP1-RNA interactions reduces mitochondrial electron transport chain activity. Hence, dAKAP1 plays a previously unappreciated role as a molecular interface between second messenger signaling and local protein synthesis machinery.

Advancing Digital Health Equity: A Policy Paper of the Infectious Diseases Society of America and the HIV Medicine Association.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic has revolutionized the practice of ambulatory medicine, triggering rapid dissemination of digital healthcare modalities, including synchronous video visits. However, social determinants of health, such as age, race, income, and others, predict readiness for telemedicine and individuals who are not able to connect virtually may become lost to care. This is particularly relevant to the practice of infectious diseases (ID) and human immunodeficiency virus (HIV) medicine, as we care for high proportions of individuals whose health outcomes are affected by such factors. Furthermore, delivering high-quality clinical care in ID and HIV practice necessitates discussion of sensitive topics, which is challenging over video without proper preparation. We describe the "digital divide," emphasize the relevance to ID and HIV practice, underscore the need to study the issue and develop interventions to mitigate its impact, and provide suggestions for optimizing telemedicine in ID and HIV clinics.

The Impact of COVID-19 on HIV Care Provided via Telemedicine-Past, Present, and Future.

PURPOSE OF REVIEW: This review summarizes HIV care delivered via telemedicine before and during the COVID-19 pandemic and highlights areas of study to inform optimal usage of telemedicine in HIV clinical practice in the future. RECENT FINDINGS: To address barriers to care created by the COVID-19 pandemic, regulatory agencies and payors waived longstanding restrictions, which enabled rapid expansion of telemedicine across the country. Preliminary data show that providers and persons with HIV (PWH) view telemedicine favorably. Some data suggest telemedicine has facilitated retention in care, but other studies have found increasing numbers of PWH lost to follow-up and worsened virologic suppression rates despite offering video and/or telephone visits. The COVID-19 pandemic has exacerbated gaps in the HIV care continuum. To help mitigate the impact, most clinics have adopted new virtual care options and are now evaluating usage, impact, and concerns. Further research into the effects of telemedicine on HIV care and continued work towards universal access are needed.

Single nucleotide polymorphisms alter kinase anchoring and the subcellular targeting of A-kinase anchoring proteins.

A-kinase anchoring proteins (AKAPs) shape second-messenger signaling responses by constraining protein kinase A (PKA) at precise intracellular locations. A defining feature of AKAPs is a helical region that binds to regulatory subunits (RII) of PKA. Mining patient-derived databases has identified 42 nonsynonymous SNPs in the PKA-anchoring helices of five AKAPs. Solid-phase RII binding assays confirmed that 21 of these amino acid substitutions disrupt PKA anchoring. The most deleterious side-chain modifications are situated toward C-termini of AKAP helices. More extensive analysis was conducted on a valine-to-methionine variant in the PKA-anchoring helix of AKAP18. Molecular modeling indicates that additional density provided by methionine at position 282 in the AKAP18γ isoform deflects the pitch of the helical anchoring surface outward by 6.6°. Fluorescence polarization measurements show that this subtle topological change reduces RII-binding affinity 8.8-fold and impairs cAMP responsive potentiation of L-type Ca2+ currents in situ. Live-cell imaging of AKAP18γ V282M-GFP adducts led to the unexpected discovery that loss of PKA anchoring promotes nuclear accumulation of this polymorphic variant. Targeting proceeds via a mechanism whereby association with the PKA holoenzyme masks a polybasic nuclear localization signal on the anchoring protein. This led to the discovery of AKAP18ε: an exclusively nuclear isoform that lacks a PKA-anchoring helix. Enzyme-mediated proximity-proteomics reveal that compartment-selective variants of AKAP18 associate with distinct binding partners. Thus, naturally occurring PKA-anchoring-defective AKAP variants not only perturb dissemination of local second-messenger responses, but also may influence the intracellular distribution of certain AKAP18 isoforms.

Depletion of dAKAP1-protein kinase A signaling islands from the outer mitochondrial membrane alters breast cancer cell metabolism and motility.

Breast cancer screening and new precision therapies have led to improved patient outcomes. Yet, a positive prognosis is less certain when primary tumors metastasize. Metastasis requires a coordinated program of cellular changes that promote increased survival, migration, and energy consumption. These pathways converge on mitochondrial function, where distinct signaling networks of kinases, phosphatases, and metabolic enzymes regulate these processes. The protein kinase A-anchoring protein dAKAP1 compartmentalizes protein kinase A (PKA) and other signaling enzymes at the outer mitochondrial membrane and thereby controls mitochondrial function and dynamics. Modulation of these processes occurs in part through regulation of dynamin-related protein 1 (Drp1). Here, we report an inverse relationship between the expression of dAKAP1 and mesenchymal markers in breast cancer. Molecular, cellular, and in silico analyses of breast cancer cell lines confirmed that dAKAP1 depletion is associated with impaired mitochondrial function and dynamics, as well as with increased glycolytic potential and invasiveness. Furthermore, disruption of dAKAP1-PKA complexes affected cell motility and mitochondrial movement toward the leading edge in invasive breast cancer cells. We therefore propose that depletion of dAKAP1-PKA "signaling islands" from the outer mitochondrial membrane augments progression toward metastatic breast cancer.

Gravin is a transitory effector of polo-like kinase 1 during cell division.

The mitogenic and second-messenger signals that promote cell proliferation often proceed through multienzyme complexes. The kinase-anchoring protein Gravin integrates cAMP and calcium/phospholipid signals at the plasma membrane by sequestering protein kinases A and C with G protein-coupled receptors. In this report we define a role for Gravin as a temporal organizer of phosphorylation-dependent protein-protein interactions during mitosis. Mass spectrometry, molecular, and cellular approaches show that CDK1/Cyclin B1 phosphorylates Gravin on threonine 766 to prime the recruitment of the polo-like kinase Plk1 at defined phases of mitosis. Fluorescent live-cell imaging reveals that cells depleted of Gravin exhibit mitotic defects that include protracted prometaphase and misalignment of chromosomes. Moreover, a Gravin T766A phosphosite mutant that is unable to interact with Plk1 negatively impacts cell proliferation. In situ detection of phospho-T766 Gravin in biopsy sections of human glioblastomas suggests that this phosphorylation event might identify malignant neoplasms.

Control of Homeostatic Synaptic Plasticity by AKAP-Anchored Kinase and Phosphatase Regulation of Ca2+-Permeable AMPA Receptors.

Neuronal information processing requires multiple forms of synaptic plasticity mediated by NMDARs and AMPA-type glutamate receptors (AMPARs). These plasticity mechanisms include long-term potentiation (LTP) and long-term depression (LTD), which are Hebbian, homosynaptic mechanisms locally regulating synaptic strength of specific inputs, and homeostatic synaptic scaling, which is a heterosynaptic mechanism globally regulating synaptic strength across all inputs. In many cases, LTP and homeostatic scaling regulate AMPAR subunit composition to increase synaptic strength via incorporation of Ca2+-permeable receptors (CP-AMPAR) containing GluA1, but lacking GluA2, subunits. Previous work by our group and others demonstrated that anchoring of the kinase PKA and the phosphatase calcineurin (CaN) to A-kinase anchoring protein (AKAP) 150 play opposing roles in regulation of GluA1 Ser845 phosphorylation and CP-AMPAR synaptic incorporation during hippocampal LTP and LTD. Here, using both male and female knock-in mice that are deficient in PKA or CaN anchoring, we show that AKAP150-anchored PKA and CaN also play novel roles in controlling CP-AMPAR synaptic incorporation during homeostatic plasticity in hippocampal neurons. We found that genetic disruption of AKAP-PKA anchoring prevented increases in Ser845 phosphorylation and CP-AMPAR synaptic recruitment during rapid homeostatic synaptic scaling-up induced by combined blockade of action potential firing and NMDAR activity. In contrast, genetic disruption of AKAP-CaN anchoring resulted in basal increases in Ser845 phosphorylation and CP-AMPAR synaptic activity that blocked subsequent scaling-up by preventing additional CP-AMPAR recruitment. Thus, the balanced, opposing phospho-regulation provided by AKAP-anchored PKA and CaN is essential for control of both Hebbian and homeostatic plasticity mechanisms that require CP-AMPARs.SIGNIFICANCE STATEMENT Neuronal circuit function is shaped by multiple forms of activity-dependent plasticity that control excitatory synaptic strength, including LTP/LTD that adjusts strength of individual synapses and homeostatic plasticity that adjusts overall strength of all synapses. Mechanisms controlling LTP/LTD and homeostatic plasticity were originally thought to be distinct; however, recent studies suggest that CP-AMPAR phosphorylation regulation is important during both LTP/LTD and homeostatic plasticity. Here we show that CP-AMPAR regulation by the kinase PKA and phosphatase CaN coanchored to the scaffold protein AKAP150, a mechanism previously implicated in LTP/LTD, is also crucial for controlling synaptic strength during homeostatic plasticity. These novel findings significantly expand our understanding of homeostatic plasticity mechanisms and further emphasize how intertwined they are with LTP and LTD.