Cytosolic Protein Kinase Research Articles

Abstract 15535: SGK1 is a Key Mediator of Pathological Cardiac Fibrosis

Background: Cardiac fibroblasts initially respond to injury adaptively, proliferating and differentiating into myofibroblasts to maintain cardiac structure and function. However, prolonged cardiac stress leads to pathological cardiac fibrosis, ultimately resulting in heart failure (HF). Thus, there is a major interest in developing therapeutic strategies to constrain fibroblast differentiation and sustained activation. We recently identified the cytosolic protein kinase, serum/glucocorticoid regulated kinase 1 (SGK1), as being largely involved with pathological remodeling in the cardiac myocyte. While there have been reports that SGK1 may also play a role in fibrosis, the mechanistic role for SGK1 in cardiac fibroblasts in response to injury remains unknown. Hypothesis: SGK1 promotes pathological cardiac fibrosis and exacerbates progression to HF. Methods: Transforming growth factor beta, (TGFβ) was used in primary cardiac fibroblasts (CFBs), in vitro , and transverse aortic constriction (TAC) was used as a HF model in mice, in vivo . CFBs were transfected with siRNA to knockdown SGK1, in vitro , and SGK1 floxed mice were injected with a novel AAV9 utilizing a myofibroblast-specific promoter to knockout SGK1 specifically in myofibroblasts (SGK1 mfbKO), in vivo . Results: SGK1 signaling was elevated in cardiac explants obtained from ischemic human HF patients which led us to further evaluate SGK1 activity in CFBs. SGK1 knockdown in CFBs attenuated TGFβ-mediated differentiation and fibrotic signaling. SGK1 mfbKO mice subjected to TAC showed preserved systolic and diastolic cardiac function, measured by echocardiography. At the biochemical level, pathological remodeling and gene markers of fibrosis were attenuated in SGK1 mfbKO mice as determined by qRT-PCR of cardiac extracts. Finally, treatment of CFBs with a small molecule SGK1 inhibitor attenuated differentiation and fibrotic signaling in response to TGFβ treatment. Conclusion: SGK1 knockdown is protective against cardiac fibrosis, attenuating differentiation and fibrotic signaling in CFBs. Further, SGK1 mfbKO protected against pathological cardiac remodeling and functional decline in a mouse model of HF, highlighting the specific pathophysiological contributions of CFBs.

Docking and Selectivity Studies of Covalently Bound Janus Kinase 3 Inhibitors.

The Janus kinases (JAKs) are a family of non-receptor cytosolic protein kinases critical for immune signaling. Many covalently bound ligands of JAK3 inhibitors have been reported. To help design selective JAK inhibitors, in this paper, we used five model proteins to study the subtype selectivity of and the mutational effects on inhibitor binding. We also compared the Covalent Dock programs from the Schrodinger software suite and the MOE software suite to determine which method to use for the drug design of covalent inhibitors. Our results showed that the docking affinity from 4Z16 (JAK3 wild-type model), 4E4N (JAK1), 4D1S (JAK2), and 7UYT (TYK2) from the Schrödinger software suite agreed well with the experimentally derived binding free energies with small predicted mean errors. However, the data from the mutant 5TTV model using the Schrödinger software suite yielded relatively large mean errors, whereas the MOE Covalent Dock program gave small mean errors in both the wild-type and mutant models for our model proteins. The docking data revealed that Leu905 of JAK3 and the hydrophobic residue at the same position in different subtypes (Leu959 of JAK1, Leu932 of JAK2, and Val981 of TYK2) is important for ligand binding to the JAK proteins. Arg911 and Asp912 of JAK3, Asp939 of JAK2, and Asp988 of TYK2 can be used for selective binding over JAK1, which contains Lys965 and Glu966 at the respective positions. Asp1021, Asp1039, and Asp1042 can be utilized for JAK1-selective ligand design, whereas Arg901 and Val981 may help guide TYK2-selective molecule design.

Tyrosine phosphorylation regulates RIPK1 activity to limit cell death and inflammation

Receptor-interacting serine/threonine-protein kinase 1 (RIPK1) is a cytosolic protein kinase that regulates multiple inflammatory and cell death pathways. Serine/Threonine phosphorylation of RIPK1 is known to suppress RIPK1 kinase-mediated cell death in the contexts of inflammation, infection and embryogenesis, however, regulation by tyrosine phosphorylation has not been reported. Here, we show that non-receptor tyrosine kinases Janus kinase 1 (JAK1) and SRC are able to phosphorylate RIPK1 at Y384 (Y383 in murine RIPK1), leading to suppression of TNF-induced cell death. Mice bearing a homozygous Ripk1 mutation that prevents tyrosine phosphorylation of RIPK1 (Ripk1Y383F/Y383F), develop systemic inflammation and emergency haematopoiesis. Mechanistically, Ripk1Y383F/Y383F mutation promotes RIPK1 kinase activation and enhances TNF-induced apoptosis and necroptosis, which is partially due to impaired recruitment and activation of MAP kinase-activated protein kinase 2 (MK2). The systemic inflammation and emergency haematopoiesis in Ripk1Y383F/Y383F mice are largely alleviated by RIPK1 kinase inhibition, and prevented by genomic deletions targeted to the upstream pathway (either to Tumor necrosis factor receptor 1 or RIPK3 and Caspase8 simultaneously). In summary, our results demonstrate that tyrosine phosphorylation of RIPK1 is critical for regulating RIPK1 activity to limit cell death and inflammation.

Noncanonical Form of ERAD Regulates Cardiac Hypertrophy.

Cardiac hypertrophy increases demands on protein folding, which causes an accumulation of misfolded proteins in the endoplasmic reticulum (ER). These misfolded proteins can be removed by the adaptive retrotranslocation, polyubiquitylation, and a proteasome-mediated degradation process, ER-associated degradation (ERAD), which, as a biological process and rate, has not been studied in vivo. To investigate a role for ERAD in a pathophysiological model, we examined the function of the functional initiator of ERAD, valosin-containing protein-interacting membrane protein (VIMP), positing that VIMP would be adaptive in pathological cardiac hypertrophy in mice. We developed a new method involving cardiac myocyte-specific adeno-associated virus serovar 9-mediated expression of the canonical ERAD substrate, TCRα, to measure the rate of ERAD, ie, ERAD flux, in the heart in vivo. Adeno-associated virus serovar 9 was also used to either knock down or overexpress VIMP in the heart. Then mice were subjected to transverse aortic constriction to induce pressure overload-induced cardiac hypertrophy. ERAD flux was slowed in both human heart failure and mice after transverse aortic constriction. Surprisingly, although VIMP adaptively contributes to ERAD in model cell lines, in the heart, VIMP knockdown increased ERAD and ameliorated transverse aortic constriction-induced cardiac hypertrophy. Coordinately, VIMP overexpression exacerbated cardiac hypertrophy, which was dependent on VIMP engaging in ERAD. Mechanistically, we found that the cytosolic protein kinase SGK1 (serum/glucocorticoid regulated kinase 1) is a major driver of pathological cardiac hypertrophy in mice subjected to transverse aortic constriction, and that VIMP knockdown decreased the levels of SGK1, which subsequently decreased cardiac pathology. We went on to show that although it is not an ER protein, and resides outside of the ER, SGK1 is degraded by ERAD in a noncanonical process we call ERAD-Out. Despite never having been in the ER, SGK1 is recognized as an ERAD substrate by the ERAD component DERLIN1, and uniquely in cardiac myocytes, VIMP displaces DERLIN1 from initiating ERAD, which decreased SGK1 degradation and promoted cardiac hypertrophy. ERAD-Out is a new preferentially favored noncanonical form of ERAD that mediates the degradation of SGK1 in cardiac myocytes, and in so doing is therefore an important determinant of how the heart responds to pathological stimuli, such as pressure overload.

Immunoproteasomes control activation of innate immune signaling and microglial function.

Microglia are the resident immune cells of the central nervous system (CNS) and play a major role in the regulation of brain homeostasis. To maintain their cellular protein homeostasis, microglia express standard proteasomes and immunoproteasomes (IP), a proteasome isoform that preserves protein homeostasis also in non-immune cells under challenging conditions. The impact of IP on microglia function in innate immunity of the CNS is however not well described. Here, we establish that IP impairment leads to proteotoxic stress and triggers the unfolded and integrated stress responses in mouse and human microglia models. Using proteomic analysis, we demonstrate that IP deficiency in microglia results in profound alterations of the ubiquitin-modified proteome among which proteins involved in the regulation of stress and immune responses. In line with this, molecular analysis revealed chronic activation of NF-κB signaling in IP-deficient microglia without further stimulus. In addition, we show that IP impairment alters microglial function based on markers for phagocytosis and motility. At the molecular level IP impairment activates interferon signaling promoted by the activation of the cytosolic stress response protein kinase R. The presented data highlight the importance of IP function for the proteostatic potential as well as for precision proteolysis to control stress and immune signaling in microglia function.

Necroptosis at a glance.

Necroptosis, or programmed necrosis, is an inflammatory form of cell death with important functions in host defense against pathogens and tissue homeostasis. The four cytosolic receptor-interacting protein kinase homotypic interaction motif (RHIM)-containing adaptor proteins RIPK1, RIPK3, TRIF (also known as TICAM1) and ZBP1 mediate necroptosis induction in response to infection and cytokine or innate immune receptor activation. Activation of the RHIM adaptors leads to phosphorylation, oligomerization and membrane targeting of the necroptosis effector protein mixed lineage kinase domain-like (MLKL). Active MLKL induces lesions on the plasma membrane, leading to the release of pro-inflammatory damage-associated molecular patterns (DAMPs). Thus, activities of the RHIM adaptors and MLKL are tightly regulated by posttranslational modifications to prevent inadvertent release of immunogenic contents. In this Cell Science at a Glance article and the accompanying poster, we provide an overview of the regulatory mechanisms of necroptosis and its biological functions in tissue homeostasis, pathogen infection and other inflammatory diseases.

TRPM7 kinase inactivation results in macrophage alkalinization and increased phagocytic activity

Transient receptor potential melastatin 7 (TRPM7) is a unique protein that is both an ion channel and a cytosolic protein kinase. Normally, TRPM7 channels are tonically inhibited by cytoplasmic Mg2+, polyamines and acidic pH. Channel basal activity can be increased by alkalinization or Mg2+ depletion. We have used the TRPM7 kinase-dead (KD K1646R) mouse to investigate the consequences of kinase inactivation in immune cell function. It also serves as a convenient system to elucidate kinase-channel interactions at a functional level.

Lights, Camera, Interaction: Studying Protein-Protein Interactions of the ER Protein Translocase in Living Cells.

Various landmark studies have revealed structures and functions of the Sec61/SecY complex in all domains of live demonstrating the conserved nature of this ancestral protein translocase. While the bacterial homolog of the Sec61 complex resides in the plasma membrane, the eukaryotic counterpart manages the transfer of precursor proteins into or across the membrane of the endoplasmic reticulum (ER). Sec61 complexes are accompanied by a set of dynamically recruited auxiliary proteins assisting the transport of certain precursor polypeptides. TRAP and Sec62/Sec63 are two auxiliary protein complexes in mammalian cells that have been characterized by structural and biochemical methods. Using these ER membrane protein complexes for our proof-of-concept study, we aimed to detect interactions of membrane proteins in living mammalian cells under physiological conditions. Bimolecular luminescence complementation and competition was used to demonstrate multiple protein–protein interactions of different topological layouts. In addition to the interaction of the soluble catalytic and regulatory subunits of the cytosolic protein kinase A, we detected interactions of ER membrane proteins that either belong to the same multimeric protein complex (intra-complex interactions: Sec61α–Sec61β, TRAPα–TRAPβ) or protein complexes in juxtaposition (inter-complex interactions: Sec61α–TRAPα, Sec61α–Sec63, and Sec61β–Sec63). In the process, we established further control elements like synthetic peptide complementation for expression profiling of fusion constructs and protease-mediated reporter degradation demonstrating the cytosolic localization of a reporter complementation. Ease of use and flexibility of the approach presented here will spur further research regarding the dynamics of protein–protein interactions in response to changing cellular conditions in living cells.

Sperm-oocyte signaling: the role of IZUMO1R and CD9 in PTK2B activation and actin remodeling at the sperm binding site†.

Sperm-oocyte binding initiates an outside-in signaling event in the mouse oocyte that triggers recruitment and activation of the cytosolic protein kinase PTK2B in the cortex underlying the bound sperm. While not involved in gamete fusion, PTK2B activity promotes actin remodeling events important during sperm incorporation. However, the mechanism by which sperm-oocyte binding activates PTK2B is unknown, and the present study examined the possibility that sperm interaction with specific oocyte surface proteins plays an important role in PTK2B activation. Imaging studies revealed that as IZUMO1R and CD9 became concentrated at the sperm binding site, activated (phosphorylated) PTK2B accumulated in the cortex underlying the sperm head and in microvilli partially encircling the sperm head. In order to determine whether IZUMO1R and/or CD9 played a significant role in PTK2B recruitment and activation at the sperm binding site, the ability of oocytes null for Izumo1r or Cd9, to initiate an increase in PTK2B content and activation was tested. The results revealed that IZUMO1R played a minor role in PTK2B activation and had no effect on actin remodeling; however, CD9 played a very significant role in PTK2B activation and subsequent actin remodeling at the sperm binding site. These findings suggest the possibility that interaction of sperm surface proteins with CD9 or CD9-associated oocyte proteins triggers PTK2B activation at the sperm binding site.

Characterizing the structure-activity relationships of natural products, tanshinones, reveals their mode of action in inhibiting spleen tyrosine kinase.

The cytosolic non-receptor protein kinase, spleen tyrosine kinase (SYK), is an attractive drug target in autoimmune, inflammatory disorder, and cancers indications. Here, we employed pharmacophore-based drug screening combined with biochemical assay and molecular dynamics (MD) simulations to identify and characterize inhibitors targeting SYK. The built pharmacophore model, phar-TanI, successfully identified tanshinone (TanI (IC50 = 1.72 μM)) and its analogs (TanIIA (IC50 = 3.2 μM), ST32da (IC50 = 46 μM), and ST32db (IC50 = 51 μM)) which apparently attenuated the activities of SYK in vitro. Additionally, the MD simulations followed by Ligplot analyses revealed that TanI and TanIIA interfered SYK activity through binding deeply into the active site. Besides, TanI and TanIIA mainly interact with residues L377, A400, V433, M448, M450, A451, E452, L453, G454, P455, and L501, which are functional hotspots for structure-based inhibitor optimization against SYK. The structure–activity relationships (SAR) study of the identified SYK inhibitors demonstrated that the pharmacophore model, phar-TanI is reliable and precise in screening inhibitors against SYK. This study disclosed the structure–function relationships of tanshinones from Traditional Chinese Medicine (Danshen), revealing their binding site and mode of action in inhibiting SYK and provides applicability in developing new therapeutic agents.

Mapping TRPM7 Function by NS8593.

The transient receptor potential cation channel, subfamily M, member 7 (TRPM7) is a ubiquitously expressed membrane protein, which forms a channel linked to a cytosolic protein kinase. Genetic inactivation of TRPM7 in animal models uncovered the critical role of TRPM7 in early embryonic development, immune responses, and the organismal balance of Zn2+, Mg2+, and Ca2+. TRPM7 emerged as a new therapeutic target because malfunctions of TRPM7 have been associated with anoxic neuronal death, tissue fibrosis, tumour progression, and giant platelet disorder. Recently, several laboratories have identified pharmacological compounds allowing to modulate either channel or kinase activity of TRPM7. Among other small molecules, NS8593 has been defined as a potent negative gating regulator of the TRPM7 channel. Consequently, several groups applied NS8593 to investigate cellular pathways regulated by TRPM7. Here, we summarize the progress in this research area. In particular, two notable milestones have been reached in the assessment of TRPM7 druggability. Firstly, several laboratories demonstrated that NS8593 treatment reliably mirrors prominent phenotypes of cells manipulated by genetic inactivation of TRPM7. Secondly, it has been shown that NS8593 allows us to probe the therapeutic potential of TRPM7 in animal models of human diseases. Collectively, these studies employing NS8593 may serve as a blueprint for the preclinical assessment of TRPM7-targeting drugs.

Neuropilin‑1 as a new potential SARS‑CoV‑2 infection mediator implicated in the neurologic features and central nervous system involvement of COVID‑19.

Infection by the severe acute respiratory syndrome (SARS) coronavirus-2 (SARS-CoV-2) is the cause of the new viral infectious disease (coronavirus disease 2019; COVID-19). Emerging evidence indicates that COVID-19 may be associated with a wide spectrum of neurological symptoms and complications with central nervous system (CNS) involvement. It is now well-established that entry of SARS-CoV-2 into host cells is facilitated by its spike proteins mainly through binding to the angiotensin-converting enzyme 2 (ACE-2). Preclinical studies have suggested that neuropilin-1 (NRP1), which is a transmembrane receptor that lacks a cytosolic protein kinase domain and exhibits high expression in the respiratory and olfactory epithelium, may also be implicated in COVID-19 by enhancing the entry of SARS-CoV-2 into the brain through the olfactory epithelium. In the present study, we expand on these findings and demonstrate that the NRP1 is also expressed in the CNS, including olfactory-related regions such as the olfactory tubercles and paraolfactory gyri. This furthers supports the potential role of NRP1 as an additional SARS-CoV-2 infection mediator implicated in the neurologic manifestations of COVID-19. Accordingly, the neurotropism of SARS-CoV-2 via NRP1-expressing cells in the CNS merits further investigation.

PIE-FLIM Measurements of Two Different FRET-Based Biosensor Activities in the Same Living Cells

We report the use of pulsed interleaved excitation (PIE)-fluorescence lifetime imaging microscopy (FLIM) to measure the activities of two different biosensor probes simultaneously in single living cells. Many genetically encoded biosensors rely on the measurement of Förster resonance energy transfer (FRET) to detect changes in biosensor conformation that accompany the targeted cell signaling event. One of the most robust ways of quantifying FRET is to measure changes in the fluorescence lifetime of the donor fluorophore using FLIM. The study of complex signaling networks in living cells demands the ability to track more than one of these cellular events at the same time. Here, we demonstrate how PIE-FLIM can separate and quantify the signals from different FRET-based biosensors to simultaneously measure changes in the activity of two cell signaling pathways in the same living cells in tissues. The imaging system described here uses selectable laser wavelengths and synchronized detection gating that can be tailored and optimized for each FRET pair. Proof-of-principle studies showing simultaneous measurement of cytosolic calcium and protein kinase A activity are shown, but the PIE-FLIM approach is broadly applicable to other signaling pathways.

A Cytosolic Protein Kinase STY46 in Arabidopsis thaliana is Involved in Plant Growth and Abiotic Stress Response.

Starch provides plants with carbon and energy during stressful periods; however, relatively few regulators of starch metabolism under stress-induced carbon starvation have been discovered. We studied a protein kinase Ser/Thr/Tyr (STY) 46, identified by gene co-expression network analysis as a potential regulator of the starch starvation response in Arabidopsis thaliana. We showed that STY46 was induced by (1) abscisic acid and prolonged darkness, (2) by abiotic stressors, including salinity and osmotic stress, and (3) by conditions associated with carbon starvation. Characterization of STY46 T-DNA knockout mutants indicated that there was functional redundancy among the STY gene family, as these genotypes did not show strong phenotypes. However, Arabidopsis with high levels of STY46 transcripts (OE-25) grew faster at the early seedling stage, had higher photosynthetic rates, and more carbon was stored as protein in the seeds under control conditions. Further, OE-25 source leaf accumulated more sugars under 100 mM NaCl stress, and salinity also accelerated root growth, which is consistent with an adaptive response. Salt-stressed OE-25 partitioned 14C towards sugars and amino acids, and away from starch and protein in source leaves. Together, these findings suggested that STY46 may be part of the salinity stress response pathway that utilizes starch during early plant growth.

Neuropilin: Handyman and Power Broker in the Tumor Microenvironment.

Neuropilin-1 and neuropilin-2 form a small family of transmembrane receptors, which, due to the lack of a cytosolic protein kinase domain, act primarily as co-receptors for various ligands. Performing at the molecular level both the executive and organizing functions of a handyman as well as of a power broker, they are instrumental in controlling the signaling of various receptor tyrosine kinases, integrins, and other molecules involved in the regulation of physiological and pathological angiogenic processes. In this setting, the various neuropilin ligands and interaction partners on various cells of the tumor microenvironment, such as cancer cells, endothelial cells, cancer-associated fibroblasts, and immune cells, are surveyed. The suitability of various neuropilin-targeting substances and the intervention in neuropilin-mediated interactions is considered as a possible building block of tumor therapy.

Neuropilins in the Context of Tumor Vasculature.

Neuropilin-1 and Neuropilin-2 form a small family of plasma membrane spanning receptors originally identified by the binding of semaphorin and vascular endothelial growth factor. Having no cytosolic protein kinase domain, they function predominantly as co-receptors of other receptors for various ligands. As such, they critically modulate the signaling of various receptor tyrosine kinases, integrins, and other molecules involved in the regulation of physiological and pathological angiogenic processes. This review highlights the diverse neuropilin ligands and interacting partners on endothelial cells, which are relevant in the context of the tumor vasculature and the tumor microenvironment. In addition to tumor cells, the latter contains cancer-associated fibroblasts, immune cells, and endothelial cells. Based on the prevalent neuropilin-mediated interactions, the suitability of various neuropilin-targeted substances for influencing tumor angiogenesis as a possible building block of a tumor therapy is discussed.

Preassembled GPCR signaling complexes mediate distinct cellular responses to ultralow ligand concentrations.

G protein-coupled receptors (GPCRs) are the largest class of cell surface signaling proteins, participate in nearly all physiological processes, and are the targets of 30% of marketed drugs. Typically, nanomolar to micromolar concentrations of ligand are used to activate GPCRs in experimental systems. We detected GPCR responses to a wide range of ligand concentrations, from attomolar to millimolar, by measuring GPCR-stimulated production of cyclic adenosine monophosphate (cAMP) with high spatial and temporal resolution. Mathematical modeling showed that femtomolar concentrations of ligand activated, on average, 40% of the cells in a population provided that a cell was activated by one to two binding events. Furthermore, activation of the endogenous β2-adrenergic receptor (β2AR) and muscarinic acetylcholine M3 receptor (M3R) by femtomolar concentrations of ligand in cell lines and human cardiac fibroblasts caused sustained increases in nuclear translocation of extracellular signal-regulated kinase (ERK) and cytosolic protein kinase C (PKC) activity, respectively. These responses were spatially and temporally distinct from those that occurred in response to higher concentrations of ligand and resulted in a distinct cellular proteomic profile. This highly sensitive signaling depended on the GPCRs forming preassembled, higher-order signaling complexes at the plasma membrane. Recognizing that GPCRs respond to ultralow concentrations of neurotransmitters and hormones challenges established paradigms of drug action and provides a previously unappreciated aspect of GPCR activation that is quite distinct from that typically observed with higher ligand concentrations.

Bcl-xL mediates RIPK3-dependent necrosis in M. tuberculosis-infected macrophages.

Virulent Mycobacterium tuberculosis (Mtb) triggers necrosis in host Mφ, which is essential for successful pathogenesis. Here we demonstrate that necrosis of Mtb-infected Mφ is dependent on the action of the cytosolic kinase Receptor Interacting Protein 3 (RIPK3) and the mitochondrial Bcl-2 family member protein B-cell lymphoma - extra large (Bcl-xL). RIPK3-deficient Mφ are able to better control bacterial growth in vitro and in vivo. Cytosolic RIPK3 translocates to the mitochondria where it promotes necrosis and blocks caspase 8-activation and apoptosis via Bcl-xL. Furthermore, necrosis is associated with stabilization of hexokinase II on the mitochondria as well as cyclophilin D-dependent mitochondrial permeability transition (MPT). These events up-regulate the level of reactive oxygen species (ROS) to induce necrosis. Thus, in Mtb-infected Mφ mitochondria are an essential platform for induction of necrosis by activating RIPK3 function and preventing caspase 8 - activation.

Light-Regulated Protein Kinases Based on the CRY2-CIB1 System.

Optogenetic approaches enable the control of biological processes in a time- and space-resolved manner. These light-based methods are noninvasive and by using light as sole activator minimize side effects in contrast to chemical inducers. Here, we provide a protocol for the targeted control of the activity of protein kinases in mammalian cells based on the photoreceptor cryptochrome 2 (CRY2) of Arabidopsis thaliana and its interaction partner CIB1. Blue light (450 nm)-induced binding of CRY2 to CIB1 allows the recruitment of a chimeric cytosolic protein kinase AKT1 to the plasma membrane accompanied with stimulation of its kinase activity. This protocol comprises the transient and stable implementation of the light-regulated system into mammalian cells and its stimulation by blue light-emitting diodes (450 nm) irradiation as well as analysis of the light-activated AKT1.

The A-Kinase Anchoring Protein (AKAP) Glycogen Synthase Kinase 3β Interaction Protein (GSKIP) Regulates β-Catenin through Its Interactions with Both Protein Kinase A (PKA) and GSK3β

The A-kinase anchoring protein (AKAP) GSK3β interaction protein (GSKIP) is a cytosolic scaffolding protein binding protein kinase A (PKA) and glycogen synthase kinase 3β (GSK3β). Here we show that both the AKAP function of GSKIP, i.e. its direct interaction with PKA, and its direct interaction with GSK3β are required for the regulation of β-catenin and thus Wnt signaling. A cytoplasmic destruction complex targets β-catenin for degradation and thus prevents Wnt signaling. Wnt signals cause β-catenin accumulation and translocation into the nucleus, where it induces Wnt target gene expression. GSKIP facilitates control of the β-catenin stabilizing phosphorylation at Ser-675 by PKA. Its interaction with GSK3β facilitates control of the destabilizing phosphorylation of β-catenin at Ser-33/Ser-37/Thr-41. The influence of GSKIP on β-catenin is explained by its scavenger function; it recruits the kinases away from the destruction complex without forming a complex with β-catenin. The regulation of β-catenin by GSKIP is specific for this AKAP as AKAP220, which also binds PKA and GSK3β, did not affect Wnt signaling. We find that the binding domain of AKAP220 for GSK3β is a conserved GSK3β interaction domain (GID), which is also present in GSKIP. Our findings highlight an essential compartmentalization of both PKA and GSK3β by GSKIP, and ascribe a function to a cytosolic AKAP-PKA interaction as a regulatory factor in the control of canonical Wnt signaling. Wnt signaling controls different biological processes, including embryonic development, cell cycle progression, glycogen metabolism, and immune regulation; deregulation is associated with diseases such as cancer, type 2 diabetes, inflammatory, and Alzheimer's and Parkinson's diseases.