Transforming Growth Factor (TGF)-β-activated Kinase 1 (TAK1) Activation Requires Phosphorylation of Serine 412 by Protein Kinase A Catalytic Subunit α (PKACα) and X-linked Protein Kinase (PRKX)

TGF-β-activated kinase 1 (TAK1) is a key kinase in mediating Toll-like receptors (TLRs) and interleukin-1 receptor (IL-1R) signaling. Although TAK1 activation involves the phosphorylation of Thr-184 and Thr-187 residues at the activation loop, the molecular mechanism underlying the complete activation of TAK1 remains elusive. In this work, we show that the Thr-187 phosphorylation of TAK1 is regulated by its C-terminal coiled-coil domain-mediated dimerization in an autophosphorylation manner. Importantly, we find that TAK1 activation in mediating downstream signaling requires an additional phosphorylation at Ser-412, which is critical for TAK1 response to proinflammatory stimuli, such as TNF-α, LPS, and IL-1β. In vitro kinase and shRNA-based knockdown assays reveal that TAK1 Ser-412 phosphorylation is regulated by cAMP-dependent protein kinase catalytic subunit α (PKACα) and X-linked protein kinase (PRKX), which is essential for proper signaling and proinflammatory cytokine induction by TLR/IL-1R activation. Morpholino-based in vivo knockdown and rescue studies show that the corresponding site Ser-391 in zebrafish TAK1 plays a conserved role in NF-κB activation. Collectively, our data unravel a previously unknown mechanism involving TAK1 phosphorylation mediated by PKACα and PRKX that contributes to innate immune signaling.     

Structure of the Nuclear Factor κB-inducing Kinase (NIK) Kinase Domain Reveals a Constitutively Active Conformation

NF-κB-inducing kinase (NIK) is a central component in the non-canonical NF-κB signaling pathway. Excessive NIK activity is implicated in various disorders, such as autoimmune conditions and cancers. Here, we report the first crystal structure of truncated human NIK in complex with adenosine 5′-O-(thiotriphosphate) at a resolution of 2.5 Å. This truncated protein is a catalytically active construct, including an N-terminal extension of 60 residues prior to the kinase domain, the kinase domain, and 20 residues afterward. The structure reveals that the NIK kinase domain assumes an active conformation in the absence of any phosphorylation. Analysis of the structure uncovers a unique role for the N-terminal extension sequence, which stabilizes helix αC in the active orientation and keeps the kinase domain in the catalytically competent conformation. Our findings shed light on the long-standing debate over whether NIK is a constitutively active kinase. They also provide a molecular basis for the recent observation of gain-of-function activity for an N-terminal deletion mutant (ΔN324) of NIK, leading to constitutive non-canonical NF-κB signaling with enhanced B-cell adhesion and apoptosis resistance.     

Mechanism of Endogenous Regulation of the Type I Interferon Response by Suppressor of IκB Kinase ? (SIKE), a Novel Substrate of TANK-binding Kinase 1 (TBK1)

TANK-binding kinase 1 (TBK1) serves as a key convergence point in multiple innate immune signaling pathways. In response to receptor-mediated pathogen detection, TBK1 phosphorylation promotes production of pro-inflammatory cytokines and type I interferons. Increasingly, TBK1 dysregulation has been linked to autoimmune disorders and cancers, heightening the need to understand the regulatory controls of TBK1 activity. Here, we describe the mechanism by which suppressor of IKKϵ (SIKE) inhibits TBK1-mediated phosphorylation of interferon regulatory factor 3 (IRF3), which is essential to type I interferon production. Kinetic analyses showed that SIKE not only inhibits IRF3 phosphorylation but is also a high affinity TBK1 substrate. With respect to IRF3 phosphorylation, SIKE functioned as a mixed-type inhibitor (K_{i, app} = 350 nm) rather than, given its status as a TBK1 substrate, as a competitive inhibitor. TBK1 phosphorylation of IRF3 and SIKE displayed negative cooperativity. Both substrates shared a similar K_m value at low substrate concentrations (∼50 nm) but deviated >8-fold at higher substrate concentrations (IRF3 = 3.5 μm; SIKE = 0.4 μm). TBK1-SIKE interactions were modulated by SIKE phosphorylation, clustered in the C-terminal portion of SIKE (Ser-133, -185, -187, -188, -190, and -198). These sites exhibited striking homology to the phosphorylation motif of IRF3. Mutagenic probing revealed that phosphorylation of Ser-185 controlled TBK1-SIKE interactions. Taken together, our studies demonstrate for the first time that SIKE functions as a TBK1 substrate and inhibits TBK1-mediated IRF3 phosphorylation by forming a high affinity TBK1-SIKE complex. These findings provide key insights into the endogenous control of a critical catalytic hub that is achieved not by direct repression of activity but by redirection of catalysis through substrate affinity.     

Phosphorylation and Inactivation of Glycogen Synthase Kinase 3β (GSK3β) by Dual-specificity Tyrosine Phosphorylation-regulated Kinase 1A (Dyrk1A)

Glycogen synthase kinase 3β (GSK3β) participates in many cellular processes, and its dysregulation has been implicated in a wide range of diseases such as obesity, type 2 diabetes, cancer, and Alzheimer disease. Inactivation of GSK3β by phosphorylation at specific residues is a primary mechanism by which this constitutively active kinase is controlled. However, the regulatory mechanism of GSK3β is not fully understood. Dual-specificity tyrosine phosphorylation-regulated kinase 1A (Dyrk1A) has multiple biological functions that occur as the result of phosphorylation of diverse proteins that are involved in metabolism, synaptic function, and neurodegeneration. Here we show that GSK3β directly interacts with and is phosphorylated by Dyrk1A. Dyrk1A-mediated phosphorylation at the Thr³⁵⁶ residue inhibits GSK3β activity. Dyrk1A transgenic (TG) mice are lean and resistant to diet-induced obesity because of reduced fat mass, which shows an inverse correlation with the effect of GSK3β on obesity. This result suggests a potential in vivo association between GSK3β and Dyrk1A regarding the mechanism underlying obesity. The level of Thr(P)³⁵⁶-GSK3β was higher in the white adipose tissue of Dyrk1A TG mice compared with control mice. GSK3β activity was differentially regulated by phosphorylation at different sites in adipose tissue depending on the type of diet the mice were fed. Furthermore, overexpression of Dyrk1A suppressed the expression of adipogenic proteins, including peroxisome proliferator-activated receptor γ, in 3T3-L1 cells and in young Dyrk1A TG mice fed a chow diet. Taken together, these results reveal a novel regulatory mechanism for GSK3β activity and indicate that overexpression of Dyrk1A may contribute to the obesity-resistant phenotype through phosphorylation and inactivation of GSK3β.     

Akt-dependent Activation of mTORC1 Complex Involves Phosphorylation of mTOR (Mammalian Target of Rapamycin) by IκB Kinase α (IKKα)

The serine/threonine protein kinase Akt promotes cell survival, growth, and proliferation through phosphorylation of different downstream substrates. A key effector of Akt is the mammalian target of rapamycin (mTOR). Akt is known to stimulate mTORC1 activity through phosphorylation of tuberous sclerosis complex 2 (TSC2) and PRAS40, both negative regulators of mTOR activity. We previously reported that IκB kinase α (IKKα), a component of the kinase complex that leads to NF-κB activation, plays an important role in promoting mTORC1 activity downstream of activated Akt. Here, we demonstrate IKKα-dependent regulation of mTORC1 using multiple PTEN null cancer cell lines and an animal model with deletion of IKKα. Importantly, IKKα is shown to phosphorylate mTOR at serine 1415 in a manner dependent on Akt to promote mTORC1 activity. These results demonstrate that IKKα is an effector of Akt in promoting mTORC1 activity.     

Phosphorylation of SU(VAR)3–9 by the Chromosomal Kinase JIL-1

The histone methyltransferase SU(VAR)3–9 plays an important role in the formation of heterochromatin within the eukaryotic nucleus. Several studies have shown that the formation of condensed chromatin is highly regulated during development, suggesting that SU(VAR)3–9''s activity is regulated as well. However, no mechanism by which this may be achieved has been reported so far. As we and others had shown previously that the N-terminus of SU(VAR)3–9 plays an important role for its activity, we purified interaction partners from Drosophila embryo nuclear extract using as bait a GST fusion protein containing the SU(VAR)3–9 N-terminus. Among several other proteins known to bind Su(VAR)3–9 we isolated the chromosomal kinase JIL-1 as a strong interactor. We show that SU(VAR)3–9 is a substrate for JIL-1 in vitro as well as in vivo and map the site of phosphorylation. These findings may provide a molecular explanation for the observed genetic interaction between SU(VAR)3–9 and JIL-1.     

Phosphorylation Sites Within α4 Subunits of α4β2 Neuronal Nicotinic Receptors: A Comparison of Substrate Specificities for cAMP-Dependent Protein Kinase (PKA) and Protein Kinase C (PKC)

The present study determined whether putative phosphorylation sites within the M3/M4 cytoplasmic domain of the human 4 subunit of 42 neuronal nicotinic receptors are substrates for cAMP-dependent protein kinase (PKA) or protein kinase C (PKC). Five peptides corresponding to predicted phosphorylation sequences were synthesized, and phosphorylation was compared with standard peptide substrates for each kinase, that is, Kemptide for PKA and glycogen synthase (GS) 1-8 for PKC. VRCRSRSI had the highest affinity for PKA, with a Km of 44.5 M; Kemptide had a Km of 7.7 M. LMKRPSVVK and KARSLSVQH were also phosphorylated by PKA, but had lower affinities of 593 M and 2896 M, respectively. LMKRPSVVK had the highest affinity for PKC with a Km of 182 M; GS 1–8 had a Km of 2.1 M. VRCRSRSI had a comparative affinity for PKC with a Km of 327 M. PCKCTCKK was not phosphorylated by PKA, but was a substrate for PKC with a Km of 1392 M, whereas PGPSCKSP was not phosphorylated by either kinase. Based on these findings, results suggest that Ser-362 and Ser-486 on the human 4 subunit may be phosphorylated by either PKA or PKC, Ser-467 is a putative PKA site, and Thr-532 represents a likely PKC substrate; Ser-421 does not appear to be phosphorylated by either kinase.     

Regulation of Macropinocytosis by Diacylglycerol Kinase ζ

Macropinosomes arise from the closure of plasma membrane ruffles to bring about the non-selective uptake of nutrients and solutes into cells. The morphological changes underlying ruffle formation and macropinosome biogenesis are driven by actin cytoskeleton rearrangements under the control of the Rho GTPase Rac1. We showed previously that Rac1 is activated by diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid. Here, we show DGKζ is required for optimal macropinocytosis induced by growth factor stimulation of mouse embryonic fibroblasts. Time-lapse imaging of live cells and quantitative analysis revealed DGKζ was associated with membrane ruffles and nascent macropinosomes. Macropinocytosis was attenuated in DGKζ-null cells, as determined by live imaging and vaccinia virus uptake experiments. Moreover, macropinosomes that did form in DGKζ-null cells were smaller than those found in wild type cells. Rescue of this defect required DGKζ catalytic activity, consistent with it also being required for Rac1 activation. A constitutively membrane bound DGKζ mutant substantially increased the size of macropinosomes and potentiated the effect of a constitutively active Rac1 mutant on macropinocytosis. Collectively, our results suggest DGKζ functions in concert with Rac1 to regulate macropinocytosis.     

Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ Isoform

Protein-protein interactions are important in providing compartmentalization and specificity in cellular signal transduction. Many studies have hallmarked the well designed compartmentalization of the cAMP-dependent protein kinase (PKA) through its anchoring proteins. Much less data are available on the compartmentalization of its closest homolog, cGMP-dependent protein kinase (PKG), via its own PKG anchoring proteins (GKAPs). For the enrichment, screening, and discovery of (novel) PKA anchoring proteins, a plethora of methodologies is available, including our previously described chemical proteomics approach based on immobilized cAMP or cGMP. Although this method was demonstrated to be effective, each immobilized cyclic nucleotide did not discriminate in the enrichment for either PKA or PKG and their secondary interactors. Hence, with PKG signaling components being less abundant in most tissues, it turned out to be challenging to enrich and identify GKAPs. Here we extend this cAMP-based chemical proteomics approach using competitive concentrations of free cyclic nucleotides to isolate each kinase and its secondary interactors. Using this approach, we identified Huntingtin-associated protein 1 (HAP1) as a putative novel GKAP. Through sequence alignment with known GKAPs and secondary structure prediction analysis, we defined a small sequence domain mediating the interaction with PKG Iβ but not PKG Iα. In vitro binding studies and site-directed mutagenesis further confirmed the specificity and affinity of HAP1 binding to the PKG Iβ N terminus. These data fully support that HAP1 is a GKAP, anchoring specifically to the cGMP-dependent protein kinase isoform Iβ, and provide further evidence that also PKG spatiotemporal signaling is largely controlled by anchoring proteins.     

Role of UDP-N-Acetylglucosamine2-Epimerase/N-Acetylmannosamine Kinase (GNE) in β1-Integrin-Mediated Cell Adhesion

Hereditary inclusion body myopathy (GNE myopathy) is a neuromuscular disorder due to mutation in key sialic acid biosynthetic enzyme, GNE. The pathomechanism of the disease is poorly understood as GNE is involved in other cellular functions beside sialic acid synthesis. In the present study, a HEK293 cell-based model system has been established where GNE is either knocked down or over-expressed along with pathologically relevant GNE mutants (D176V and V572L). The subcellular distribution of recombinant GNE and its mutant showed differential localization in the cell. The effect of mutation on GNE function was investigated by studying hyposialylation of cell membrane receptor, β1-integrin. Hyposialylated β1-integrin localized to internal vesicles that was restored upon supplementation with sialic acid. Fibronectin stimulation caused migration of hyposialylated β1-integrin to the cell membrane and co-localization with focal adhesion kinase (FAK) leading to increased focal adhesion formation. This further activated FAK and Src, downstream signaling molecules and led to increased cell adhesion. This is the first report to show that mutation in GNE affects β1-integrin-mediated cell adhesion process in GNE mutant cells.     

Grb2 Promotes Integrin-Induced Focal Adhesion Kinase (FAK) Autophosphorylation and Directs the Phosphorylation of Protein Tyrosine Phosphatase α by the Src-FAK Kinase Complex

The integrin-activated Src-focal adhesion kinase (FAK) kinase complex phosphorylates PTPα at Tyr789, initiating PTPα-mediated signaling that promotes cell migration. Recruitment of the BCAR3-Cas complex by PTPα-phospho-Tyr789 at focal adhesions is one mechanism of PTPα signaling. The adaptor protein Grb2 is also recruited by PTPα-phospho-Tyr789, although the role of the PTPα-Grb2 complex in integrin signaling is unknown. We show that silencing Grb2 expression in fibroblasts abolishes PTPα-Tyr789 phosphorylation and that this is due to two unexpected actions of Grb2. First, Grb2 promotes integrin-induced autophosphorylation of FAK-Tyr397. This is impaired in Grb2-depleted cells and prohibits FAK activation and formation of the Src-FAK complex. Grb2-depleted cells contain less paxillin, and paxillin overexpression rescues FAK-Tyr397 phosphorylation, suggesting that the FAK-activating action of Grb2 involves paxillin. A second distinct role for Grb2 in PTPα-Tyr789 phosphorylation involves Grb2-mediated coupling of Src-FAK and PTPα. This requires two phosphosites, FAK-Tyr925 and PTPα-Tyr789, for Grb2-Src homology 2 (SH2) binding. We propose that a Grb2 dimer links FAK and PTPα, and this positions active Src-FAK in proximity with other, perhaps integrin-clustered, molecules of PTPα to enable maximal PTPα-Tyr789 phosphorylation. These findings identify Grb2 as a new FAK activator and reveal its essential role in coordinating PTPα tyrosine phosphorylation to enable downstream integrin signaling and migration.     

Cell Surface Expression of the Major Amyloid-β Peptide (Aβ)-degrading Enzyme, Neprilysin, Depends on Phosphorylation by Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase Kinase (MEK) and Dephosphorylation by Protein Phosphatase 1a

Neprilysin is one of the major amyloid-β peptide (Aβ)-degrading enzymes, the expression of which declines in the brain during aging. The decrease in neprilysin leads to a metabolic Aβ imbalance, which can induce the amyloidosis underlying Alzheimer disease. Pharmacological activation of neprilysin during aging therefore represents a potential strategy to prevent the development of Alzheimer disease. However, the regulatory mechanisms mediating neprilysin activity in the brain remain unclear. To address this issue, we screened for pharmacological regulators of neprilysin activity and found that the neurotrophic factors brain-derived neurotrophic factor, nerve growth factor, and neurotrophins 3 and 4 reduce cell surface neprilysin activity. This decrease was mediated by MEK/ERK signaling, which enhanced phosphorylation at serine 6 in the neprilysin intracellular domain (S6-NEP-ICD). Increased phosphorylation of S6-NEP-ICD in primary neurons reduced the levels of cell surface neprilysin and led to a subsequent increase in extracellular Aβ levels. Furthermore, a specific inhibitor of protein phosphatase-1a, tautomycetin, induced extensive phosphorylation of the S6-NEP-ICD, resulting in reduced cell surface neprilysin activity. In contrast, activation of protein phosphatase-1a increased cell surface neprilysin activity and lowered Aβ levels. Taken together, these results indicate that the phosphorylation status of S6-NEP-ICD influences the localization of neprilysin and affects extracellular Aβ levels. Therefore, maintaining S6-NEP-ICD in a dephosphorylated state, either by inhibition of protein kinases involved in its phosphorylation or by activation of phosphatases catalyzing its dephosphorylation, may represent a new approach to prevent reduction of cell surface neprilysin activity during aging and to maintain physiological levels of Aβ in the brain.     

Carboxyl Terminus of HSC70-interacting Protein (CHIP) Down-regulates NF-κB-inducing Kinase (NIK) and Suppresses NIK-induced Liver Injury

Ser/Thr kinase NIK (NF-κB-inducing kinase) mediates the activation of the noncanonical NF-κB2 pathway, and it plays an important role in regulating immune cell development and liver homeostasis. NIK levels are extremely low in quiescent cells due to ubiquitin/proteasome-mediated degradation, and cytokines stimulate NIK activation through increasing NIK stability; however, regulation of NIK stability is not fully understood. Here we identified CHIP (carboxyl terminus of HSC70-interacting protein) as a new negative regulator of NIK. CHIP contains three N-terminal tetratricopeptide repeats (TPRs), a middle dimerization domain, and a C-terminal U-box. The U-box domain contains ubiquitin E3 ligase activity that promotes ubiquitination of CHIP-bound partners. We observed that CHIP bound to NIK via its TPR domain. In both HEK293 and primary hepatocytes, overexpression of CHIP markedly decreased NIK levels at least in part through increasing ubiquitination and degradation of NIK. Accordingly, CHIP suppressed NIK-induced activation of the noncanonical NF-κB2 pathway. CHIP also bound to TRAF3, and CHIP and TRAF3 acted coordinately to efficiently promote NIK degradation. The TPR but not the U-box domain was required for CHIP to promote NIK degradation. In mice, hepatocyte-specific overexpression of NIK resulted in liver inflammation and injury, leading to death, and liver-specific expression of CHIP reversed the detrimental effects of hepatic NIK. Our data suggest that CHIP/TRAF3/NIK interactions recruit NIK to E3 ligase complexes for ubiquitination and degradation, thus maintaining NIK at low levels. Defects in CHIP regulation of NIK may result in aberrant NIK activation in the liver, contributing to live injury, inflammation, and disease.     

Cyclic AMP- and (Rp)-cAMPS-induced Conformational Changes in a Complex of the Catalytic and Regulatory (RIα) Subunits of Cyclic AMP-dependent Protein Kinase

We took a discovery approach to explore the actions of cAMP and two of its analogs, one a cAMP mimic ((S_p)-adenosine cyclic 3′:5′-monophosphorothioate ((S_p)-cAMPS)) and the other a diastereoisomeric antagonist ((R_p)-cAMPS), on a model system of the type Iα cyclic AMP-dependent protein kinase holoenzyme, RIα(91–244)·C-subunit, by using fluorescence spectroscopy and amide H/²H exchange mass spectrometry. Specifically, for the fluorescence experiments, fluorescein maleimide was conjugated to three cysteine single residue substitution mutants, R92C, T104C, and R239C, of RIα(91–244), and the effects of cAMP, (S_p)-cAMPS, and (R_p)-cAMPS on the kinetics of R-C binding and the time-resolved anisotropy of the reporter group at each conjugation site were measured. For the amide exchange experiments, ESI-TOF mass spectrometry with pepsin proteolytic fragmentation was used to assess the effects of (R_p)-cAMPS on amide exchange of the RIα(91–244)·C-subunit complex. We found that cAMP and its mimic perturbed at least parts of the C-subunit interaction Sites 2 and 3 but probably not Site 1 via reduced interactions of the linker region and αC of RIα(91–244). Surprisingly, (R_p)-cAMPS not only increased the affinity of RIα(91–244) toward the C-subunit by 5-fold but also produced long range effects that propagated through both the C- and R-subunits to produce limited unfolding and/or enhanced conformational flexibility. This combination of effects is consistent with (R_p)-cAMPS acting by enhancing the internal entropy of the R·C complex. Finally, the (R_p)-cAMPS-induced increase in affinity of RIα(91–244) toward the C-subunit indicates that (R_p)-cAMPS is better described as an inverse agonist because it decreases the fractional dissociation of the cyclic AMP-dependent protein kinase holoenzyme and in turn its basal activity.Cyclic AMP-dependent protein kinase (PKA)¹ plays a crucial role in a plethora of cellular functions. All isoforms of PKA are composed of two catalytic (C) subunits and homodimeric regulatory (R) subunits (1–3). As the name implies, cAMP is a major PKA regulator (4). Much progress has been made in the last decade in delineating the molecular basis of action of cAMP. An important tactic in this endeavor has been through the comparison of the effects of cAMP with those of two phosphorothioate cAMP analogs: (S_p)-cAMPS (a cAMP mimic) and (R_p)-cAMPS (an antagonist and a diastereoisomer of (S_p)-cAMPS). Although the importance of geometry of the sulfur substitution is critical in determining the pharmacological properties of the two phosphorothioate cAMP analogs, the molecular basis for this behavior is not fully understood. To date, these comparisons have only been made using either wild-type or truncated mutants of the type Iα regulatory subunit (RIα) that are free in solution, not complexed to the C-subunit. X-ray spectroscopic examination of ligand-bound RIα(92–379) complexes reveals few differences between ligand-bound complexes, but the (R_p)-cAMPS complex is structurally “looser” with higher thermal factors than complexes formed with either cAMP or (S_p)-cAMPS (5). This is consistent with the observation that both cAMP and (S_p)-cAMPS, but not (R_p)-cAMPS, raise the urea concentration required for wild-type RIα unfolding (6). Further insight into the structural basis of cAMP action stems from NMR spectroscopic comparison of the effects of (R_p)-cAMPS, cAMP, and (S_p)-cAMPS on chemical shifts and ¹⁵N relaxation of the RIα(119–244) mutant (7). In addition to producing fewer significant chemical shift changes than either cAMP or (S_p)-cAMPS, (R_p)-cAMPS binding is associated with enhanced millisecond to microsecond time scale backbone motions of a β-turn (β2,3 loop) and around the phosphate-binding cassette (PBC) (7).Further insight into the molecular basis of actions of cAMP and its analogs should come from the analysis of ligand-bound R·C complexes. Unfortunately, the large size of even the heterodimeric R·C complex (∼95 kDa) and the difficulty of preparing (R_p)-cAMPS·R·C-subunit crystals currently preclude the use of both NMR spectroscopy and x-ray crystallography. Consequently, we took two alternative lower resolution approaches to this issue. One approach involves the use of site-directed labeling combined with fluorescence spectroscopy to examine both the effects of cAMP and its analogs on R-C subunit binding kinetics and on the conformational dynamics of RIα(91–244). RIα(91–244) includes the “A” cyclic nucleotide binding (CNB) domain, the pseudosubstrate, and linker domains and represents the minimal segments necessary for high affinity C-subunit binding (Fig. 1) (8). The other approach involves an examination of the effects of cAMP and its analogs on solvent exposure/conformational flexibility of RIα(91–244)·C-subunit complex using H/²H amide exchange measured with a combination of mass spectrometry (ESI-Q-TOF) and proteolytic fragmentation. In the first approach, fluorescein maleimide (FM) was conjugated to three cysteine substitution mutants with the substitution sites located near or within the pseudosubstrate sequence, the linker domain, or αC (R92C, T104C, and R239C, respectively) of RIα(91–244) (Fig. 1). The time-resolved fluorescence anisotropy results suggest that cAMP and (S_p)-cAMPS reduce the interaction of the RIα linker domain and αC with the two peripheral R-C interaction sites on the C-subunit (so-called Sites 2 and 3) without affecting the interaction of the pseudosubstrate sequence with the active site cleft (so-called Site 1). Because of limitations of the amide H/²H exchange experiments, only the effects of (R_p)-cAMPS on H/²H amide exchange in RIα(91–244)·C-subunit complex could be investigated. The results showed that (R_p)-cAMPS induces a relatively widespread increase in amide exchange, indicating limited unfolding and/or enhanced conformational flexibility that is propagated almost globally through the C-subunit and, at least, part of RIα. These conformational changes were accompanied by a 5-fold increase in the affinity of RIα(91–244) toward C-subunit, suggesting that, at least, some of the (R_p)-cAMPS effects are mediated by an increase in internal entropy. Finally, the (R_p)-cAMPS-induced increase in R-C affinity indicates that (R_p)-cAMPS is better described as an inverse agonist because the basal activity of the PKA holoenzyme should be decreased by (R_p)-cAMPS.Open in a separate windowFig. 1.Overview of PKA structure and cAMP analogs. A, domain organization of RIα showing the domain boundaries of RIα(91–244) where the pseudosubstrate in green is connected to CNB-A domain in blue by a linker segment. B, structure of RIα(91–244) in the C-subunit-bound conformation (Protein Data Bank code 1U7E (23)) showing the pseudosubstrate in green, linker in yellow, and helical subdomain comprising helices αN, αA, αB, and αC in blue and β-subdomain in tan. The PBC is in red. C, structure of the C·RIα(91–244) holoenzyme showing the C-subunit in tan and RIα(91–244) in blue. Sites for introduction of cysteines by site-directed mutagenesis are represented by red circles. The cAMP binding site (PBC) is in red. D, structure of cAMP showing the 2′-OH group and 3′–5′ phosphodiester bond. The exocyclic oxygens upon replacement with sulfur atoms to generate the (S_p)-cAMPS and (R_p)-cAMPS diastereomers are highlighted.     

5′-AMP-activated Protein Kinase (AMPK) Supports the Growth of Aggressive Experimental Human Breast Cancer Tumors

Rapid tumor growth can establish metabolically stressed microenvironments that activate 5′-AMP-activated protein kinase (AMPK), a ubiquitous regulator of ATP homeostasis. Previously, we investigated the importance of AMPK for the growth of experimental tumors prepared from HRAS-transformed mouse embryo fibroblasts and for primary brain tumor development in a rat model of neurocarcinogenesis. Here, we used triple-negative human breast cancer cells in which AMPK activity had been knocked down to investigate the contribution of AMPK to experimental tumor growth and core glucose metabolism. We found that AMPK supports the growth of fast-growing orthotopic tumors prepared from MDA-MB-231 and DU4475 breast cancer cells but had no effect on the proliferation or survival of these cells in culture. We used in vitro and in vivo metabolic profiling with [¹³C]glucose tracers to investigate the contribution of AMPK to core glucose metabolism in MDA-MB-231 cells, which have a Warburg metabolic phenotype; these experiments indicated that AMPK supports tumor glucose metabolism in part through positive regulation of glycolysis and the nonoxidative pentose phosphate cycle. We also found that AMPK activity in the MDA-MB-231 tumors could systemically perturb glucose homeostasis in sensitive normal tissues (liver and pancreas). Overall, our findings suggest that the contribution of AMPK to the growth of aggressive experimental tumors has a critical microenvironmental component that involves specific regulation of core glucose metabolism.     

Glycogen Synthase Kinase 3 Beta (GSK3β) at the Tip of Neuronal Development and Regeneration

Gaining a basic understanding of the inhibitory molecules and the intracellular signaling involved in axon development and repulsion after neural lesions is of clear biomedical interest. In recent years, numerous studies have described new molecules and intracellular mechanisms that impair axonal outgrowth after injury. In this scenario, the role of glycogen synthase kinase 3 beta (GSK3β) in the axonal responses that occur after central nervous system (CNS) lesions began to be elucidated. GSK3β function in the nervous tissue is associated with neural development, neuron polarization, and, more recently, neurodegeneration. In fact, GSK3β has been considered as a putative therapeutic target for promoting functional recovery in injured or degenerative CNS. In this review, we summarize current understanding of the role of GSK3β during neuronal development and regeneration. In particular, we discuss GSK3β activity levels and their possible impact on cytoskeleton dynamics during both processes.     

β1 Integrin-Focal Adhesion Kinase (FAK) Signaling Modulates Retinal Ganglion Cell (RGC) Survival

Extracellular matrix (ECM) integrity in the central nervous system (CNS) is essential for neuronal homeostasis. Signals from the ECM are transmitted to neurons through integrins, a family of cell surface receptors that mediate cell attachment to ECM. We have previously established a causal link between the activation of the matrix metalloproteinase-9 (MMP-9), degradation of laminin in the ECM of retinal ganglion cells (RGCs), and RGC death in a mouse model of retinal ischemia-reperfusion injury (RIRI). Here we investigated the role of laminin-integrin signaling in RGC survival in vitro, and after ischemia in vivo. In purified primary rat RGCs, stimulation of the β1 integrin receptor with laminin, or agonist antibodies enhanced RGC survival in correlation with activation of β1 integrin’s major downstream regulator, focal adhesion kinase (FAK). Furthermore, β1 integrin binding and FAK activation were required for RGCs’ survival response to laminin. Finally, in vivo after RIRI, we observed an up-regulation of MMP-9, proteolytic degradation of laminin, decreased RGC expression of β1 integrin, FAK and Akt dephosphorylation, and reduced expression of the pro-survival molecule bcl-xL in the period preceding RGC apoptosis. RGC death was prevented, in the context of laminin degradation, by maintaining β1 integrin activation with agonist antibodies. Thus, disruption of homeostatic RGC-laminin interaction and signaling leads to cell death after retinal ischemia, and maintaining integrin activation may be a therapeutic approach to neuroprotection.