Phosphorylation-independent Regulation of Metabotropic Glutamate Receptor 5 Desensitization and Internalization by G Protein-coupled Receptor Kinase 2 in Neurons

The uncoupling of metabotropic glutamate receptors (mGluRs) from heterotrimeric G proteins represents an essential feedback mechanism that protects neurons against receptor overstimulation that may ultimately result in damage. The desensitization of mGluR signaling is mediated by both second messenger-dependent protein kinases and G protein-coupled receptor kinases (GRKs). Unlike mGluR1, the attenuation of mGluR5 signaling in HEK 293 cells is reported to be mediated by a phosphorylation-dependent mechanism. However, the mechanisms regulating mGluR5 signaling and endocytosis in neurons have not been investigated. Here we show that a 2-fold overexpression of GRK2 leads to the attenuation of endogenous mGluR5-mediated inositol phosphate (InsP) formation in striatal neurons and siRNA knockdown of GRK2 expression leads to enhanced mGluR5-mediated InsP formation. Expression of a catalytically inactive GRK2-K220R mutant also effectively attenuates mGluR5 signaling, but the expression of a GRK2-D110A mutant devoid in Gα_q/11 binding increases mGluR5 signaling in response to agonist stimulation. Taken together, these results indicate that the attenuation of mGluR5 responses in striatal neurons is phosphorylation-independent. In addition, we find that mGluR5 does not internalize in response to agonist treatment in striatal neuron, but is efficiently internalized in cortical neurons that have higher levels of endogenous GRK2 protein expression. When overexpressed in striatal neurons, GRK2 promotes agonist-stimulated mGluR5 internalization. Moreover, GRK2-mediated promotion of mGluR5 endocytosis does not require GRK2 catalytic activity. Thus, we provide evidence that GRK2 mediates phosphorylation-independent mGluR5 desensitization and internalization in neurons.Glutamate is the major excitatory neurotransmitter in the mammalian brain and functions to activate two distinct classes of receptors (ionotropic and metabotropic) to regulate a variety of physiological functions (1–3). Ionotropic glutamate receptors, such as NMDA, AMPA, and kainate receptors, are ligand-gated ion channels, whereas metabotropic glutamate receptors (mGluRs)⁵ are members of the G protein-coupled receptor (GPCR) superfamily (4–7). mGluRs modulate synaptic activity via the activation of heterotrimeric G proteins that are coupled to a variety of second messenger cascades. Group I mGluRs (mGluR1 and mGluR5) are coupled to the activation of Gα_q/11 proteins, which stimulate the activation of phospholipase Cβ1 resulting in diacylglycerol (DAG) and inositol-1,4,5-trisphosphate (IP₃) formation, release of Ca²⁺ from intracellular stores and subsequent activation of protein kinase C.The attenuation of GPCR signaling is mediated in part by G protein-coupled receptor kinases (GRKs), which phosphorylate GPCRs to promote the binding of β-arrestin proteins that uncouple GPCRs from heterotrimeric G proteins (8–10). GRK2 has been demonstrated to contribute to the phosphorylation and desensitization of both mGluR1 and mGluR5 in human embryonic kidney (HEK 293) cells (11–17). GRK4 is also implicated in mediating the desensitization of mGluR1 signaling in cerebellar Purkinje cells, but does not contribute to the desensitization of mGluR5 (14, 15). In addition, GRK4 plays a major role in mGluR1 internalization (13, 14). A role for GRK2 in promoting mGluR1 internalization is less clear as different laboratories have obtained discordant results (11, 14, 15, 16). However, the only study examining the role of GRK2 in regulating mGluR1 endocytosis in a native system reported that GRK2 knockdown had no effect upon mGluR1 internalization in cerebellar Purkinje cells (14).GRK2 is composed of three functional domains: an N-terminal regulator of G protein signaling (RGS) homology (RH) domain, a central catalytic domain, and a C-terminal G_βγ binding pleckstrin homology domain (18). In HEK 293 cells, mGluR1 desensitization is not dependent on GRK2 catalytic activity. Rather the GRK2 RH domain interacts with both the second intracellular loop domain of mGluR1 and the α-subunit of Gα_q/11 and attenuates second messenger responses by disrupting the mGluR1/Gα_q/11 signaling complexes (12, 19–21). Although the molecular mechanism underlying GRK2-mediated attenuation of mGluR1 signaling is relatively well established in HEK 293 cells, the role of GRK2 in regulating the desensitization of mGluRs in neurons remains to be determined. Moreover, it is not known whether GRK2-dependent attenuation of mGluR5 signaling is mediated by the same phosphorylation-independent mechanism that has been described for mGluR1. In a previous study, GRK2-mediated mGluR5 desensitization was reported to be phosphorylation-dependent, based on the observation that the overexpression of a catalytically inactive GRK2 (K220R) did not attenuate mGluR5 signaling (15). In the present study, we examined whether a 2-fold overexpression of GRK2 in primary mouse striatal neurons to match GRK2 expression levels found in the cortex results in increased agonist-stimulated desensitization and internalization of endogenous mGluR5. We report here that GRK2 mediates phosphorylation-independent mGluR5 desensitization and internalization. Furthermore, GRK2 knockdown causes an increase in mGluR5 signaling, demonstrating that endogenous GRK2 plays a role in mGluR5 desensitization.     

G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

During conditions of moderate sodium excess, the dopaminergic system regulates blood pressure and water and electrolyte balance by engendering natriuresis. Dopamine exerts its effects on dopamine receptors, including the dopamine D₃ receptor. G protein-coupled receptor kinase 4 (GRK4), whose gene locus (4p16.3) is linked to essential hypertension, desensitizes the D₁ receptor, another dopamine receptor. This study evaluated the role of GRK4 on D₃ receptor function in human proximal tubule cells. D₃ receptor co-segregated in lipid rafts and co-immunoprecipitated and co-localized in human proximal tubule cells and in proximal and distal tubules and glomeruli of kidneys of Wistar Kyoto rats. Bimolecular fluorescence complementation and confocal microscopy revealed that agonist activation of the receptor initiated the interaction between D₃ receptor and GRK4 at the cell membrane and promoted it intracellularly, presumably en route to endosomal trafficking. Of the four GRK4 splice variants, GRK4-γ and GRK4-α mediated a 3- and 2-fold increase in the phosphorylation of agonist-activated D₃ receptor, respectively. Inhibition of GRK activity with heparin or knockdown of GRK4 expression via RNA interference completely abolished p44/42 phosphorylation and mitogenesis induced by D₃ receptor stimulation. These data demonstrate that GRK4, specifically the GRK4-γ and GRK4-α isoforms, phosphorylates the D₃ receptor and is crucial for its signaling in human proximal tubule cells.During conditions of moderate sodium excess, the dopaminergic system sits at the fulcrum of homeostatic control of water and electrolyte balance and blood pressure (1, 2). Dopamine promotes natriuresis by inhibiting sodium chloride reabsorption in specific segments of the nephron. Dopamine exerts its action on dopamine receptors, which belong to the family of G protein-coupled receptors (GPCRs).² The dopamine receptors are classified into two subtypes based on their ability to increase cAMP levels, sequence similarity, G protein coupling, and pharmacological profiles (3, 4). The D₁-like dopamine receptors activate adenylyl cyclase by coupling to stimulatory Gα_s/Gα_olf and include the D₁ (D₁R) and D₅ receptors (D₅R). The D₂-like dopamine receptors inhibit adenylyl cyclase by coupling to Gα_i/Gα_o and consist of the D₂ (D₂R), D₃ (D₃R), and D₄ (D₄R) receptors. The D₃R has also been shown to couple to Gα_o, Gβγ, and to the stimulatory Gα_s (5, 6).The signal transduction that follows ligand occupation of a GPCR is tightly regulated to limit the specificity and extent of cellular response. GPCR-mediated signal transduction is rapidly dampened via receptor desensitization or the waning of the responsiveness of the receptor to agonist with time. Desensitization involves receptor phosphorylation and is carried out by either GPCR kinases (GRKs) or second messenger-activated kinases such as protein kinase A and protein kinase C. Homologous desensitization involves GRKs that selectively phosphorylate only agonist-activated receptors, whereas heterologous desensitization is carried out by second messenger-dependent kinases that indiscriminately phosphorylate agonist-activated receptors and those that have not been exposed to the agonist (7).The GRKs are serine/threonine protein kinases comprising seven isoforms that are grouped into three subfamilies. GRK1 and GRK7 belong to the rhodopsin kinase subfamily and are expressed exclusively in the retina (8–10). GRK2 and GRK3 phosphorylate the β-adrenergic receptor and belong to the β-adrenergic receptor kinase subfamily (11), and GRK4, GRK5, and GRK6 belong to the GRK4 subfamily. GRK4 is highly enriched in the testis and, to a lesser degree, in the kidneys (12, 13). Four splice variants of human GRK4 result from the alternative splicing of exons 2 and 15 (11). GRK4-α is considered the full-length version, whereas GRK4-β, -γ, and -δ are shortened versions of GRK4-α (14). The coding region of the GRK4 gene, whose 4p16.3 locus has been linked to essential hypertension (15, 16), contains several single nucleotide polymorphisms, including R65L, A142V, and A486V, which have been linked to essential hypertension and/or salt sensitivity in various ethnic groups (17).The D₃R gene is found at 3q13.3 (18), a locus that is also linked to essential hypertension (19, 20). Sequence analysis of the D₃R gene shows the presence of several single nucleotide polymorphisms, which do not correlate with either essential hypertension among Japanese (21) or with blood pressure levels and diabetic nephropathy among Finns (22). However, D₃R knock-out mice develop a renin-dependent form of hypertension and fail to excrete a sodium load (23).The D₃R has a long third intracellular loop that contains several putative GRK phosphorylation sites (24). A previous study evaluated the ability of GRK2 and GRK3 to phosphorylate D₃R and showed that co-transfection of GRK3, but not GRK2, resulted in a weak phosphorylation of the heterologously expressed, dopamine-stimulated D₃R in HEK-293 (25), a human embryonic kidney cell line. We tested the hypothesis that GRK4 is required in D₃R signaling in terminally differentiated human renal proximal tubule cells (hPTCs) by determining the spatiotemporal dynamics of the interaction of D₃R and GRK4 through their subfractionation in membrane microdomains and subcellular co-localization via confocal microscopy and bimolecular fluorescence complementation assay (BiFC). We also identified which of the GRK4 splice variants are involved in D₃R phosphorylation and evaluated the physiological roles of GRK4 in D₃R signaling in the hPTCs. We now report that D₃R and GRK4 co-fractionate in lipid rafts and co-localize in both hPTCs and WYK kidneys. Moreover, D₃R is phosphorylated by GRK4-γ and GRK4-α isoforms, and the absence of GRK4 impairs D₃R-mediated mitogenesis and activation of p44/42 in hPTCs.     

GRK5 Deficiency Leads to Reduced Hippocampal Acetylcholine Level via Impaired Presynaptic M2/M4 Autoreceptor Desensitization

G protein-coupled receptor kinase 5 (GRK5) deficiency has been linked recently to early Alzheimer disease (AD), but the mechanism by which GRK5 deficiency may contribute to AD pathogenesis remains elusive. Here we report that overexpression of dominant negative mutant of GRK5 (dnGRK5) in a cholinergic neuronal cell line led to decreased acetylcholine (ACh) release. This reduction was fully corrected by pertussis toxin, atropine (a nonselective muscarinic antagonist), or methoctramine (a selective M2/M4 muscarinic receptor antagonist). Consistent with results in cultured cells, high potassium-evoked ACh release in hippocampal slices from young GRK5 knock-out mice was significantly reduced compared with wild type littermates, and this reduced ACh release was also fully corrected by methoctramine. In addition, following treatment with the nonselective muscarinic agonist oxotremorine-M, M2, and M4 receptors underwent significantly reduced internalization in GRK5KO slices compared with wild type slices, as assessed by plasma membrane retention of receptor immunoreactivity, whereas M1 receptor internalization was not affected by loss of GRK5 expression. Moreover, Western blotting revealed no synaptic or cholinergic degenerative changes in young GRK5 knock-out mice. Altogether, these results suggest that GRK5 deficiency leads to a reduced hippocampal ACh release and cholinergic hypofunction by selective impairment of desensitization of presynaptic M2/M4 autoreceptors. Because this nonstructural cholinergic hypofunction precedes the hippocampal cholinergic hypofunction associated with structural cholinergic degeneration and cognitive decline in aged GRK5 knock-out mice, this nonstructural alteration may be an early event contributing to cholinergic degeneration in AD.G protein-coupled receptor kinase-5 (GRK5)² is one of the seven GRK family members whose primary function is to desensitize G protein-coupled receptors (GPCRs) (1, 2). We recently reported that increased soluble β-amyloid decreases membrane (functional) levels of GRK5 in vitro, and this membrane GRK5 deficiency occurs in vivo as well in an Alzheimer disease (AD) transgenic model (3) and in postmortem human AD brain samples (4). Moreover, the aged GRK5 knock-out (GRK5KO) mouse, which models this GRK5 deficiency in the absence of exogenous mutant human β-amyloid precursor protein (β-APP) or any other known AD-related genes (i.e. presenilins or tau), develops axonal defects and mild cholinergic degeneration with associated amnestic mild cognitive impairment (5). When Swedish mutant βAPP is overexpressed in the GRK5KO mice by cross-breeding with Swedish APP transgenic mice, the aged double mutant mice display significantly exaggerated brain inflammation (6). These accumulating data strongly suggest that GRK5 deficiency significantly contributes to AD pathogenesis, although the precise molecular mechanisms remain to be delineated.Mounting evidence indicates that the substrate spectrum of broadly expressed GRKs (i.e. GRK2/3/5/6) can significantly overlap for some receptors, suggesting that a lack of one of these members may have only a limited impact on GPCR regulation (2). On the other hand, compensation for loss of a particular GRK member by others in vivo can be incomplete or selective for other receptor types. For example, GRK2KO and GRK6KO mice have been shown to display selective impairments of adrenergic and dopaminergic receptor desensitization, respectively (7, 8). Findings from different GRK isoform-targeted animals strongly support the conclusion that although redundancy exists between GRK isoforms, each isoform has its own selective substrates; should one GRK be deficient or inactivated, desensitization of its selective substrates will be impaired (1). For GRK5 in particular, previous studies have demonstrated that GRK5KO mice display selectively impaired desensitization of muscarinic acetylcholine receptors (mAChRs) (9, 10).To date, five mAChR subtypes have been identified, with M1, M3, and M5 receptors being G_q/11-coupled, and M2 and M4 receptors being G_i/o-coupled (11). In hippocampal memory circuits, M2 receptor (M2R) is primarily a presynaptic autoreceptor that inhibits ACh release (12, 13), whereas M1R is postsynaptic and is believed to be critical in memory processes involving an interaction between the cerebral cortex and hippocampus (11). In AD, there is a selective loss of cholinergic neurons that leads to a cholinergic hypofunction, primarily a hypoactivity of postsynaptic nicotinic and M1 muscarinic receptors (14). GRK5KO mice, when challenged with nonselective muscarinic agonists, display augmented hypothermia, hypoactivity, tremor, and salivation, as well as antinociceptive changes (9). These behavioral changes are typical M2 and/or M4 receptor-mediated functions, according to the findings from muscarinic receptor subtype knock-out mice (11, 15). Therefore, GRK5 deficiency in vivo may selectively impair M2/M4R desensitization. If so, the resulting presynaptic M2/M4R hyperactivity would overly inhibit ACh release from cholinergic neurons and eventually compromise the learning and memory function. This study was undertaken to investigate the impact of GRK5 deficiency on ACh release and desensitization of mAChR subtypes using GRK5-deficient models both in vitro and in hippocampal slices from the GRK5KO mice.     

Role of Partitioning-defective 1/Microtubule Affinity-regulating Kinases in the Morphogenetic Activity of Helicobacter pylori CagA

Helicobacter pylori CagA plays a key role in gastric carcinogenesis. Upon delivery into gastric epithelial cells, CagA binds and deregulates SHP-2 phosphatase, a bona fide oncoprotein, thereby causing sustained ERK activation and impaired focal adhesions. CagA also binds and inhibits PAR1b/MARK2, one of the four members of the PAR1 family of kinases, to elicit epithelial polarity defect. In nonpolarized gastric epithelial cells, CagA induces the hummingbird phenotype, an extremely elongated cell shape characterized by a rear retraction defect. This morphological change is dependent on CagA-deregulated SHP-2 and is thus thought to reflect the oncogenic potential of CagA. In this study, we investigated the role of the PAR1 family of kinases in the hummingbird phenotype. We found that CagA binds not only PAR1b but also other PAR1 isoforms, with order of strength as follows: PAR1b > PAR1d ≥ PAR1a > PAR1c. Binding of CagA with PAR1 isoforms inhibits the kinase activity. This abolishes the ability of PAR1 to destabilize microtubules and thereby promotes disassembly of focal adhesions, which contributes to the hummingbird phenotype. Consistently, PAR1 knockdown potentiates induction of the hummingbird phenotype by CagA. The morphogenetic activity of CagA was also found to be augmented through inhibition of non-muscle myosin II. Because myosin II is functionally associated with PAR1, perturbation of PAR1-regulated myosin II by CagA may underlie the defect of rear retraction in the hummingbird phenotype. Our findings reveal that CagA systemically inhibits PAR1 family kinases and indicate that malfunctioning of microtubules and myosin II by CagA-mediated PAR1 inhibition cooperates with deregulated SHP-2 in the morphogenetic activity of CagA.Infection with Helicobacter pylori strains bearing cagA (cytotoxin-associated gene A)-positive strains is the strongest risk factor for the development of gastric carcinoma, the second leading cause of cancer-related death worldwide (1–3). The cagA gene is located within a 40-kb DNA fragment, termed the cag pathogenicity island, which is specifically present in the genome of cagA-positive H. pylori strains (4–6). In addition to cagA, there are ∼30 genes in the cag pathogenicity island, many of which encode a bacterial type IV secretion system that delivers the cagA-encoded CagA protein into gastric epithelial cells (7–10). Upon delivery into gastric epithelial cells, CagA is localized to the plasma membrane, where it undergoes tyrosine phosphorylation at the C-terminal Glu-Pro-Ile-Tyr-Ala motifs by Src family kinases or c-Abl kinase (11–14). The C-terminal Glu-Pro-Ile-Tyr-Ala-containing region of CagA is noted for the structural diversity among distinct H. pylori isolates. Oncogenic potential of CagA has recently been confirmed by a study showing that systemic expression of CagA in mice induces gastrointestinal and hematological malignancies (15).When expressed in gastric epithelial cells, CagA induces morphological transformation termed the hummingbird phenotype, which is characterized by the development of one or two long and thin protrusions resembling the beak of the hummingbird. It has been thought that the hummingbird phenotype is related to the oncogenic action of CagA (7, 16–19). Pathophysiological relevance for the hummingbird phenotype in gastric carcinogenesis has recently been provided by the observation that infection with H. pylori carrying CagA with greater ability to induce the hummingbird phenotype is more closely associated with gastric carcinoma (20–23). Elevated motility of hummingbird cells (cells showing the hummingbird phenotype) may also contribute to invasion and metastasis of gastric carcinoma.In host cells, CagA interacts with the SHP-2 phosphatase, C-terminal Src kinase, and Crk adaptor in a tyrosine phosphorylation-dependent manner (16, 24, 25) and also associates with Grb2 adaptor and c-Met in a phosphorylation-independent manner (26, 27). Among these CagA targets, much attention has been focused on SHP-2 because the phosphatase has been recognized as a bona fide oncoprotein, gain-of-function mutations of which are found in various human malignancies (17, 18, 28). Stable interaction of CagA with SHP-2 requires CagA dimerization, which is mediated by a 16-amino acid CagA-multimerization (CM)² sequence present in the C-terminal region of CagA (29). Upon complex formation, CagA aberrantly activates SHP-2 and thereby elicits sustained ERK MAP kinase activation that promotes mitogenesis (30). Also, CagA-activated SHP-2 dephosphorylates and inhibits focal adhesion kinase (FAK), causing impaired focal adhesions. It has been shown previously that both aberrant ERK activation and FAK inhibition by CagA-deregulated SHP-2 are involved in induction of the hummingbird phenotype (31).Partitioning-defective 1 (PAR1)/microtubule affinity-regulating kinase (MARK) is an evolutionally conserved serine/threonine kinase originally isolated in C. elegans (32–34). Mammalian cells possess four structurally related PAR1 isoforms, PAR1a/MARK3, PAR1b/MARK2, PAR1c/MARK1, and PAR1d/MARK4 (35–37). Among these, PAR1a, PAR1b, and PAR1c are expressed in a variety of cells, whereas PAR1d is predominantly expressed in neural cells (35, 37). These PAR1 isoforms phosphorylate microtubule-associated proteins (MAPs) and thereby destabilize microtubules (35, 38), allowing asymmetric distribution of molecules that are involved in the establishment and maintenance of cell polarity.In polarized epithelial cells, CagA disrupts the tight junctions and causes loss of apical-basolateral polarity (39, 40). This CagA activity involves the interaction of CagA with PAR1b/MARK2 (19, 41). CagA directly binds to the kinase domain of PAR1b in a tyrosine phosphorylation-independent manner and inhibits the kinase activity. Notably, CagA binds to PAR1b via the CM sequence (19). Because PAR1b is present as a dimer in cells (42), CagA may passively homodimerize upon complex formation with the PAR1 dimer via the CM sequence, and this PAR1-directed CagA dimer would form a stable complex with SHP-2 through its two SH2 domains.Because of the critical role of CagA in gastric carcinogenesis (7, 16–19), it is important to elucidate the molecular basis underlying the morphogenetic activity of CagA. In this study, we investigated the role of PAR1 isoforms in induction of the hummingbird phenotype by CagA, and we obtained evidence that CagA-mediated inhibition of PAR1 kinases contributes to the development of the morphological change by perturbing microtubules and non-muscle myosin II.     

Homo-oligomerization and Activation of AMP-activated Protein Kinase Are Mediated by the Kinase Domain ��G-Helix

AMP-activated protein kinase (AMPK) is a heterotrimeric complex playing a crucial role in maintaining cellular energy homeostasis. Recently, homodimerization of mammalian AMPK and yeast ortholog SNF1 was shown by us and others. In SNF1, it involved specific hydrophobic residues in the kinase domain αG-helix. Mutation of the corresponding AMPK α-subunit residues (Val-219 and Phe-223) to glutamate reduced the tendency of the kinase to form higher order homo-oligomers, as was determined by the following three independent techniques in vitro: (i) small angle x-ray scattering, (ii) surface plasmon resonance spectroscopy, and (iii) two-dimensional blue native/SDS-PAGE. Recombinant protein as well as AMPK in cell lysates of primary cells revealed distinct complexes of various sizes. In particular, the assembly of very high molecular mass complexes was dependent on both the αG-helix-mediated hydrophobic interactions and kinase activation. In vitro and when overexpressed in double knock-out (α1^−/−, α2^−/−) mouse embryonic fibroblast cells, activation of mutant AMPK was impaired, indicating a critical role of the αG-helix residues for AMPK activation via its upstream kinases. Also inactivation by protein phosphatase 2Cα was affected in mutant AMPK. Importantly, activation of mutant AMPK by LKB1 was restored by exchanging the corresponding and conserved hydrophobic αG-helix residues of LKB1 (Ile-260 and Phe-264) to positively charged amino acids. These mutations functionally rescued LKB1-dependent activation of mutant AMPK in vitro and in cell culture. Our data suggest a physiological role for the hydrophobic αG-helix residues in homo-oligomerization of heterotrimers and cellular interactions, in particular with upstream kinases, indicating an additional level of AMPK regulation.The maintenance of energy homeostasis is a basic requirement of all living organisms. The AMP-activated protein kinase (AMPK)² is crucially involved in this essential process by playing a central role in sensing and regulating energy metabolism on the cellular and whole body level (1–6). AMPK is also participating in several signaling pathways associated with cancer and metabolic diseases, like type 2 diabetes mellitus, obesity, and other metabolic disorders (7–9).Mammalian AMPK belongs to a highly conserved family of serine/threonine protein kinases with homologs found in all eukaryotic organisms examined (1, 3, 10). Its heterotrimeric structure includes a catalytic α-subunit and regulatory β- and γ-subunits. These subunits exist in different isoforms (α1, α2, β1, β2, γ1, γ2, and γ3) and splice variants (for γ2 and γ3) and can thus assemble to a broad variety of heterotrimeric isoform combinations. The α- and β-subunits possess multiple autophosphorylation sites, which have been implicated in regulation of subcellular localization and kinase activation (11–15). The most critical step of AMPK activation, however, is phosphorylation of Thr-172 within the activation segment of the α-subunit kinase domain. At least two AMPK upstream kinases (AMPKKs) have been identified so far, namely the tumor suppressor kinase LKB1 in complex with MO25 and STRAD (16) and Ca²⁺/calmodulin-dependent protein kinase kinase-2 (CamKK2) (17). Furthermore, the transforming growth factor-β-activated kinase 1 was also shown to activate AMPK using a variety of in vitro approaches (18), but the physiological relevance of these findings remains unclear. Besides direct phosphorylation of Thr-172, AMPK activity is stimulated by the allosteric activator AMP, which can bind to two Bateman domains formed by two pairs of CBS domains within the γ-subunit (19–22). Hereby bound AMP not only allosterically stimulates AMPK but also protects Thr-172 from dephosphorylation by protein phosphatase 2Cα (PP2Cα) and thus hinders inactivation of the kinase (19, 22, 23). Consequently, on the cellular level, AMPK is activated upon metabolic stress increasing the AMP/ATP ratio. Furthermore, AMPK activation can also be induced by several chemical compounds, like nucleoside 5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside (24) and the anti-diabetic drug Metformin (25–28). In addition, the small molecule compound A-769662 was recently developed as a direct allosteric activator of AMPK (29, 30).Previous work in our groups proposed a model of AMPK regulation by AMP, which incorporates the major functional features and the latest structural information (31). The latter mainly included truncated core complexes of AMPK from different species (32–35). Further valuable structural information is provided by the x-ray structures of the isolated catalytic domains, in particular of the human AMPK α2-subunit (Protein Data Bank code 2H6D) and its yeast ortholog SNF1 (36, 37). The kinase domain of SNF1 is capable of forming homodimers in the protein crystal, as well as in vitro in solution, in a unique way, which has not been observed previously in any other kinase (36). The dimer interface is predominantly formed by hydrophobic interactions of the loop-αG region, also known as subdomain X situated on the large kinase lobe (36, 38, 39), and it mainly involves Ile-257 and Phe-261. Because the T-loop activation segment was buried within the dimer interface, it was suggested that the dimeric state of the SNF1 catalytic domain represents the inactive form of the kinase. Intriguingly, it was shown in our groups by small angle x-ray scattering that AMPK self-organizes in a concentration-dependent manner to form homo-oligomers in solution (31). However, the interface responsible for oligomerization of the AMPK heterotrimer has remained elusive.Here we further investigate the distinct oligomeric states of the AMPK heterotrimer and suggest a possible regulatory function for this process. Most importantly, we provide conclusive evidence for participation of αG-helix residues in the recognition of AMPK by its upstream kinases LKB1 and CamKK2.     

Proinsulin and the Genetics of Diabetes Mellitus

Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.Insulin plays a central role in the regulation of vertebrate metabolism. The hormone, the post-translational product of a single-chain precursor, is a globular protein containing two chains, A (21 residues) and B (30 residues). Recent advances in human genetics have identified dominant mutations in the insulin gene causing permanent neonatal-onset DM² (1–4). The mutations are predicted to block folding of the precursor in the ER of pancreatic β-cells. Although expression of the wild-type allele would in other circumstances be sufficient to maintain homeostasis, studies of a corresponding mouse model (5–7) suggest that the misfolded variant perturbs wild-type biosynthesis (8, 9). Impaired β-cell secretion is associated with ER stress, distorted organelle architecture, and cell death (10). These findings have renewed interest in insulin biosynthesis (11–13) and the structural basis of disulfide pairing (14–19). Protein evolution is constrained not only by structure and function but also by susceptibility to toxic misfolding.     

The Kinase Activity of Rip2 Determines Its Stability and Consequently Nod1- and Nod2-mediated Immune Responses

Rip2 (RICK, CARD3) has been identified as a key effector molecule downstream of the pattern recognition receptors, Nod1 and Nod2; however, its mechanism of action remains to be elucidated. In particular, it is unclear whether its kinase activity is required for signaling or for maintaining protein stability. We have investigated the expression level of different retrovirally expressed kinase-dead Rip2 mutants and the role of Rip2 kinase activity in the signaling events that follow Nod1 and Nod2 stimulation. We show that in primary cells expressing kinase-inactive Rip2, protein levels were severely compromised, and stability could not be reconstituted by the addition of a phospho-mimetic mutation in its autophosphorylation site. Consequently, inflammatory cytokine production in response to Nod1 and Nod2 ligands was abrogated both in vitro and in vivo in the absence of Rip2 kinase activity. Our results highlight the central role that Rip2 kinase activity plays in conferring stability to the protein and thus in the preservation of Nod1- and Nod2-mediated innate immune responses.A key step in the initiation of effector immune responses is the recognition of highly conserved molecules expressed by microbial pathogens. The immune system has developed specific receptors that sense these so-called pathogen-associated molecular patterns and initiate appropriate immune responses. One key family of pattern recognition receptors is the Nod-like receptor (NLR)² family (1–3), of which two members, Nod1 and Nod2, have been implicated in the recognition of bacterial peptidoglycan derivatives released into the cytosol upon bacterial infection (4–6). Several studies have shown that Nod1 plays a role in host defense against invasive pathogens such as Helicobacter pylori and Escherichia coli (7, 8), and Nod2 mutations have been associated with a higher incidence of Crohn disease (9, 10), thus highlighting these NLRs as important regulators of inflammatory immune responses.Rip2, also called CARD3, RICK, or CARDIAK, is a serine/threonine kinase, which was implicated in the induction of NF-κB activation and apoptosis (11–13). Rip2 has been described to be critical for responses against Toll-like receptor ligands such as LPS (14, 15), although findings from recent studies did not support this conclusion (16). Rip2 contains a caspase-recruitment domain (CARD), which mediates interaction with other CARD-containing proteins such as Nod1 and Nod2, in addition to an N-terminal kinase domain and an intermediate domain. Nod1 and Nod2 associate with Rip2 upon peptidoglycan ligation (17) leading to downstream signaling events that culminate in NF-κB and mitogen-activated protein kinase activation (15, 18–20). Recent reports have suggested that the mitogen-activated protein kinase kinase kinase family member TAK1 provides the link between Rip2 and NF-κB activation upon Nod1 and Nod2 stimulation (21–23). However, the exact role of Rip2 and in particular its kinase activity in mediating downstream effector activation in NLR signaling still remains unclear. Notably, in vitro investigations have suggested that Rip2 kinase activity may be dispensable for the induction of immune responses initiated by NLR-ligands (21, 24, 25) and that disruption of Rip2 kinase activity is associated with a loss in protein stability (23); however, such studies utilized protein overexpression in cell lines and are yet to be tested in primary cells or in vivo.In the current investigation we sought to elucidate the role of Rip2 kinase activity in transducing inflammatory signals upon NLR stimulation in vitro and in vivo. To this end, we utilized both Rip2 knock-out (15) and Rip2 kinase-dead knock-in mice (24) in addition to Rip2 deficient primary cells that were retrovirally reconstituted with different kinase-inactive mutants. We show here that in the absence of intact kinase activity, Rip2 protein is not stable and that insertion of a phospho-mimetic mutation is not sufficient to restore stability. Moreover, pharmacological abrogation of Rip2 kinase activity in primary cells similarly leads to destabilization of the molecule. As a consequence, signaling downstream of Nod1 and Nod2 and inflammatory cytokine production is impaired both in vivo and in vitro. Our results highlight Rip2 kinase activity as a central regulator of protein stability and consequently innate immune responses triggered by Nod1 and Nod2 ligands.     

��-Defensins in Enteric Innate Immunity: FUNCTIONAL PANETH CELL ��-DEFENSINS IN MOUSE COLONIC LUMEN*

Paneth cells are a secretory epithelial lineage that release dense core granules rich in host defense peptides and proteins from the base of small intestinal crypts. Enteric α-defensins, termed cryptdins (Crps) in mice, are highly abundant in Paneth cell secretions and inherently resistant to proteolysis. Accordingly, we tested the hypothesis that enteric α-defensins of Paneth cell origin persist in a functional state in the mouse large bowel lumen. To test this idea, putative Crps purified from mouse distal colonic lumen were characterized biochemically and assayed in vitro for bactericidal peptide activities. The peptides comigrated with cryptdin control peptides in acid-urea-PAGE and SDS-PAGE, providing identification as putative Crps. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry experiments showed that the molecular masses of the putative α-defensins matched those of the six most abundant known Crps, as well as N-terminally truncated forms of each, and that the peptides contain six Cys residues, consistent with identities as α-defensins. N-terminal sequencing definitively revealed peptides with N termini corresponding to full-length, (des-Leu)-truncated, and (des-Leu-Arg)-truncated N termini of Crps 1–4 and 6. Crps from mouse large bowel lumen were bactericidal in the low micromolar range. Thus, Paneth cell α-defensins secreted into the small intestinal lumen persist as intact and functional forms throughout the intestinal tract, suggesting that the peptides may mediate enteric innate immunity in the colonic lumen, far from their upstream point of secretion in small intestinal crypts.Antimicrobial peptides (AMPs)² are released by epithelial cells onto mucosal surfaces as effectors of innate immunity (1–5). In mammals, most AMPs derive from two major families, the cathelicidins and defensins (6). The defensins comprise the α-, β-, and θ-defensin subfamilies, which are defined by the presence of six cysteine residues paired in characteristic tridisulfide arrays (7). α-Defensins are highly abundant in two primary cell lineages: phagocytic leukocytes, primarily neutrophils, of myeloid origin and Paneth cells, which are secretory epithelial cells located at the base of the crypts of Lieberkühn in the small intestine (8–10). Neutrophil α-defensins are stored in azurophilic granules and contribute to non-oxidative microbial cell killing in phagolysosomes (11, 12), except in mice whose neutrophils lack defensins (13). In the small bowel, α-defensins and other host defense proteins (14–18) are released apically as components of Paneth cell secretory granules in response to cholinergic stimulation and after exposure to bacterial antigens (19). Therefore, the release of Paneth cell products into the crypt lumen is inferred to protect mitotically active crypt cells from colonization by potential pathogens and confer protection against enteric infection (7, 20, 21).Under normal, homeostatic conditions, Paneth cells are not found outside the small bowel, although they may appear ectopically in response to local inflammation throughout the gastrointestinal tract (22, 23). Paneth cell numbers increase progressively throughout the small intestine, occurring at highest numbers in the distal ileum (24). Mouse Paneth cells express numerous α-defensin isoforms, termed cryptdins (Crps) (25), that have broad spectrum antimicrobial activities (6, 26). Collectively, α-defensins constitute approximately seventy percent of the bactericidal peptide activity in mouse Paneth cell secretions (19), selectively killing bacteria by membrane-disruptive mechanisms (27–30). The role of Paneth cell α-defensins in gastrointestinal mucosal immunity is evident from studies of mice transgenic for human enteric α-defensin-5, HD-5, which are immune to infection by orally administered Salmonella enterica sv. typhimurium (S. typhimurium) (31).The biosynthesis of mature, bactericidal α-defensins from their inactive precursors requires activation by lineage-specific proteolytic convertases. In mouse Paneth cells, inactive ∼8.4-kDa Crp precursors are processed intracellularly into microbicidal ∼4-kDa Crps by specific cleavage events mediated by matrix metalloproteinase-7 (MMP-7) (32, 33). MMP-7 null mice exhibit increased susceptibility to systemic S. typhimurium infection and decreased clearance of orally administered non-invasive Escherichia coli (19, 32). Although the α-defensin proregions are sensitive to proteolysis, the mature, disulfide-stabilized peptides resist digestion by their converting enzymes in vitro, whether the convertase is MMP-7 (32), trypsin (34), or neutrophil serine proteinases (35). Because α-defensins resist proteolysis in vitro, we hypothesized that Paneth cell α-defensins resist degradation and remain in a functional state in the large bowel, a complex, hostile environment containing varied proteases of both host and microbial origin.Here, we report on the isolation and characterization of a population of enteric α-defensins from the mouse colonic lumen. Full-length and N-terminally truncated Paneth cell α-defensins were identified and are abundant in the distal large bowel lumen.     

Phosphorylation Dependence of Hsp27 Multimeric Size and Molecular Chaperone Function

The molecular chaperone Hsp27 exists as a distribution of large oligomers that are disassembled by phosphorylation at Ser-15, -78, and -82. It is controversial whether the unphosphorylated Hsp27 or the widely used triple Ser-to-Asp phospho-mimic mutant is the more active molecular chaperone in vitro. This question was investigated here by correlating chaperone activity, as measured by the aggregation of reduced insulin or α-lactalbumin, with Hsp27 self-association as monitored by analytical ultracentrifugation. Furthermore, because the phospho-mimic is generally assumed to reproduce the phosphorylated molecule, the size and chaperone activity of phosphorylated Hsp27 were compared with that of the phospho-mimic. Hsp27 was triply phosphorylated by MAPKAP-2 kinase, and phosphorylation was tracked by urea-PAGE. An increasing degree of suppression of insulin or α-lactalbumin aggregation correlated with a decreasing Hsp27 self-association, which was the least for phosphorylated Hsp27 followed by the mimic followed by the unphosphorylated protein. It was also found that Hsp27 added to pre-aggregated insulin did not reverse aggregation but did inhibit these aggregates from assembling into even larger aggregates. This chaperone activity appears to be independent of Hsp27 phosphorylation. In conclusion, the most active chaperone of insulin and α-lactalbumin was the Hsp27 (elongated) dimer, the smallest Hsp27 subunit observed under physiological conditions. Next, the Hsp27 phospho-mimic is only a partial mimic of phosphorylated Hsp27, both in self-association and in chaperone function. Finally, the efficient inhibition of insulin aggregation by Hsp27 dimer led to the proposal of two models for this chaperone activity.Oligomeric heat shock protein 27 (Hsp27)² is a ubiquitous mammalian protein with a variety of functions in health and disease (1–8). These functions include ATP-independent chaperone activity in response to environmental stress, e.g. heat shock and oxidative stress, control of apoptosis, and regulation of actin cytoskeleton dynamics. Hsp27 is a member of the α-crystallin small heat shock protein family of which αB-crystallin is the archetype. These proteins are characterized by an α-crystallin domain of 80–90 residues consisting of roughly eight β-strands that form an intermolecular β-sheet interaction interface within a dimer, the basic building subunit of the oligomer (2, 4, 9–11).Hsp27 is in equilibrium between high molecular weight oligomers and much lower molecular weight multimers. It has been reported that unphosphorylated Hsp27 includes predominantly a distribution of high molecular species ranging in size from 12-mer to 35-mer (12–19). Phosphorylation of Hsp27 at serines 15, 78, and 82 by the p38-activated MAPKAP-2 kinase (20–22) or the use of the triple Ser-to-Asp phospho-mimic results in a major shift in the equilibrium toward much smaller multimers (23) and in an alteration of its function (1, 3, 6, 7, 24, 25). The size distribution of the smaller species has been reported to be between monomer and tetramer (12–16, 18, 19).Small heat shock proteins, including Hsp27, behave as ATP-independent molecular chaperones during cellular heat shock. They bind partially unfolded proteins and prevent their aggregation until the proteins can be refolded by larger ATP-dependent chaperones or are digested (7, 8, 26). This function includes the up-regulation and/or phosphorylation of Hsp27.It is not entirely clear what the role of Hsp27 size and phosphorylation state plays in its heat shock function because there are conflicting results in the literature. Some in vitro studies concluded that the unphosphorylated oligomeric Hsp27 (or the murine isoform Hsp25) protects proteins against aggregation better than does the phosphorylation mimic (13, 19, 27), whereas others found no difference (16, 28, 29), and still other studies found that the mimic protects better than does the unphosphorylated wild type (27, 30, 31). In-cell studies found that phosphorylation of Hsp27 was essential for thermo-protection of actin filaments (32), and the Hsp27 phosphorylation mimic decreased inclusion body formation better than did unphosphorylated Hsp27 (33). This study was undertaken to investigate the molecular chaperone function of Hsp27 by correlating chaperone activity with Hsp27 size and by comparing fully phosphorylated Hsp27 with its phospho-mimic.     

Dbf4-Cdc7 Phosphorylation of Mcm2 Is Required for Cell Growth

The Dbf4-Cdc7 kinase (DDK) is required for the activation of the origins of replication, and DDK phosphorylates Mcm2 in vitro. We find that budding yeast Cdc7 alone exists in solution as a weakly active multimer. Dbf4 forms a likely heterodimer with Cdc7, and this species phosphorylates Mcm2 with substantially higher specific activity. Dbf4 alone binds tightly to Mcm2, whereas Cdc7 alone binds weakly to Mcm2, suggesting that Dbf4 recruits Cdc7 to phosphorylate Mcm2. DDK phosphorylates two serine residues of Mcm2 near the N terminus of the protein, Ser-164 and Ser-170. Expression of mcm2-S170A is lethal to yeast cells that lack endogenous MCM2 (mcm2Δ); however, this lethality is rescued in cells harboring the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Mcm2 is required for cell growth.The Cdc7 protein kinase is required throughout the yeast S phase to activate origins (1, 2). The S phase cyclin-dependent kinase also activates yeast origins of replication (3–5). It has been proposed that Dbf4 activates Cdc7 kinase in S phase, and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6). However, it is not known how Dbf4-Cdc7 (DDK)² acts during S phase to trigger the initiation of DNA replication. DDK has homologs in other eukaryotic species, and the role of Cdc7 in activation of replication origins during S phase may be conserved (7–10).The Mcm2-7 complex functions with Cdc45 and GINS to unwind DNA at a replication fork (11–15). A mutation of MCM5 (mcm5-bob1) bypasses the cellular requirements for DBF4 and CDC7 (16), suggesting a critical physiologic interaction between Dbf4-Cdc7 and Mcm proteins. DDK phosphorylates Mcm2 in vitro with proteins purified from budding yeast (17, 18) or human cells (19). Furthermore, there are mutants of MCM2 that show synthetic lethality with DBF4 mutants (6, 17), suggesting a biologically relevant interaction between DBF4 and MCM2. Nevertheless, the physiologic role of DDK phosphorylation of Mcm2 is a matter of dispute. In human cells, replacement of MCM2 DDK-phosphoacceptor residues with alanines inhibits DNA replication, suggesting that Dbf4-Cdc7 phosphorylation of Mcm2 in humans is important for DNA replication (20). In contrast, mutation of putative DDK phosphorylation sites at the N terminus of Schizosaccharomyces pombe Mcm2 results in viable cells, suggesting that phosphorylation of S. pombe Mcm2 by DDK is not critical for cell growth (10).In budding yeast, Cdc7 is present at high levels in G₁ and S phase, whereas Dbf4 levels peak in S phase (18, 21, 22). Furthermore, budding yeast DDK binds to chromatin during S phase (6), and it has been shown that Dbf4 is required for Cdc7 binding to chromatin in budding yeast (23, 24), fission yeast (25), and Xenopus (9). Human and fission yeast Cdc7 are inert on their own (7, 8), but Dbf4-Cdc7 is active in phosphorylating Mcm proteins in budding yeast (6, 26), fission yeast (7), and human (8, 10). Based on these data, it has been proposed that Dbf4 activates Cdc7 kinase in S phase and that Dbf4 interaction with Cdc7 is essential for Cdc7 kinase activity (6, 9, 18, 21–24). However, a mechanistic analysis of how Dbf4 activates Cdc7 has not yet been accomplished. For example, the multimeric state of the active Dbf4-Cdc7 complex is currently disputed. A heterodimer of fission yeast Cdc7 (Hsk1) in complex with fission yeast Dbf4 (Dfp1) can phosphorylate Mcm2 (7). However, in budding yeast, oligomers of Cdc7 exist in the cell (27), and Dbf4-Cdc7 exists as oligomers of 180 and 300 kDa (27).DDK phosphorylates the N termini of human Mcm2 (19, 20, 28), human Mcm4 (10), budding yeast Mcm4 (26), and fission yeast Mcm6 (10). Although the sequences of the Mcm N termini are poorly conserved, the DDK sites identified in each study have neighboring acidic residues. The residues of budding yeast Mcm2 that are phosphorylated by DDK have not yet been identified.In this study, we find that budding yeast Cdc7 is weakly active as a multimer in phosphorylating Mcm2. However, a low molecular weight form of Dbf4-Cdc7, likely a heterodimer, has a higher specific activity for phosphorylation of Mcm2. Dbf4 or DDK, but not Cdc7, binds tightly to Mcm2, suggesting that Dbf4 recruits Cdc7 to Mcm2. DDK phosphorylates two serine residues of Mcm2, Ser-164 and Ser-170, in an acidic region of the protein. Mutation of Ser-170 is lethal to yeast cells, but this phenotype is rescued by the DDK bypass mutant mcm5-bob1. We conclude that DDK phosphorylation of Ser-170 of Mcm2 is required for budding yeast growth.     

Inflammasome-dependent Caspase-1 Activation in Cervical Epithelial Cells Stimulates Growth of the Intracellular Pathogen Chlamydia trachomatis

Inflammasomes have been extensively characterized in monocytes and macrophages, but not in epithelial cells, which are the preferred host cells for many pathogens. Here we show that cervical epithelial cells express a functional inflammasome. Infection of the cells by Chlamydia trachomatis leads to activation of caspase-1, through a process requiring the NOD-like receptor family member NLRP3 and the inflammasome adaptor protein ASC. Secretion of newly synthesized virulence proteins from the chlamydial vacuole through a type III secretion apparatus results in efflux of K⁺ through glibenclamide-sensitive K⁺ channels, which in turn stimulates production of reactive oxygen species. Elevated levels of reactive oxygen species are responsible for NLRP3-dependent caspase-1 activation in the infected cells. In monocytes and macrophages, caspase-1 is involved in processing and secretion of pro-inflammatory cytokines such as interleukin-1β. However, in epithelial cells, which are not known to secrete large quantities of interleukin-1β, caspase-1 has been shown previously to enhance lipid metabolism. Here we show that, in cervical epithelial cells, caspase-1 activation is required for optimal growth of the intracellular chlamydiae.Chlamydia trachomatis is the most common cause of bacterial sexually transmitted disease in the United States, and it is the leading cause of preventable blindness in the world (1–5). Untreated, C. trachomatis infection in women can cause pelvic inflammatory disease, which can lead to infertility and ectopic pregnancy because of scarring of the ovaries and the Fallopian tubes (6). Infection by the lymphogranuloma venereum (LGV)² strain of C. trachomatis, which has become more common in North America and Europe (7, 8), is characterized by swelling and inflammation of the lymph nodes in the groin (9).Chlamydiae are intracellular pathogens that preferentially infect epithelial mucosa and have a biphasic infection cycle (10). A metabolically inactive form, the elementary body, infects the epithelial host cells through entry vesicles that avoid fusion with host cell lysosomes and develop into a membrane-bound inclusion (11–13). Despite their intravacuolar localization, chlamydiae are still able to acquire nutrients from the host cell and interact with host-cell signaling pathways (13–23). Within a few hours, the elementary bodies differentiate into larger, metabolically active reticulate bodies, which proliferate but are noninfectious. Depending on the strain of C. trachomatis, the reticulate bodies transform back into elementary bodies after 1–3 days and are released into the extracellular medium to infect other cells (11, 24, 25). Chlamydial species possess a type III secretion (T3S) system that secretes bacterial virulence factors into host cell cytosol and may control interactions between the inclusion and host-cell compartments (26).Long before the adaptive immune response is activated, infected epithelial cells produce proinflammatory cytokines and chemokines, including interleukin (IL)-6, IL-8, and granulocyte-macrophage colony-stimulating factor (27), which recruit neutrophils to the site of infection and activate other immune effector cells. However, in many cases the immune system fails to clear the infection, and the chronic release of cytokines becomes a major contributor to the scarring and damage associated with the infection (28–30).The innate immune response during C. trachomatis infection is initiated by chlamydial pathogen-associated molecular patterns, including lipopolysaccharides, which bind to pattern recognition receptors such as Toll-like receptors and cytosolic NOD-like receptors (NLRs), ultimately promoting pro-inflammatory cytokine gene expression and secretion of the cytokine proteins (31–37). However, secretion of the key pro-inflammatory cytokine IL-1β is tightly regulated (38). First, pro-IL-1β is produced following activation of pattern recognition receptor, and the precursor is then cleaved into the mature form by the pro-inflammatory cysteine protease, caspase-1 (also known as interleukin-1 converting enzyme or ICE). The mechanism by which caspase-1 is activated in response to infection or tissue damage was found to be modulated by a macromolecular protein complex termed the “inflammasome,” which consists of an NLR family member, an adaptor protein (apoptosis-associated speck-like protein containing a caspase activation recruitment domain or ASC), and an inactive caspase-1 precursor (pro-caspase-1) (39, 40). Previous studies demonstrated that IL-1β is produced in response to chlamydial infection in dendritic cells, macrophages, and monocytes (41–44). Moreover, C. trachomatis or Chlamydia caviae infection activates caspase-1 in epithelial cells or monocytes (43, 45, 46). However, whether caspase-1 activation during chlamydial infection requires the formation of an inflammasome remains unclear.Previous studies have shown that different pathogens can cause inflammasome-mediated caspase-1 activation in macrophages and monocytes (47). However, epithelial cells lining mucosal surfaces are not only the preferred target for chlamydial infection and other intracellular pathogens but also play an important role in early host immune response to infection by secreting proinflammatory cytokines and chemokines (27). Although epithelial cells are not known to secrete large amounts of IL-1β, inflammasome-dependent caspase-1 activation in epithelial cells is known to contribute to lipid metabolism and membrane regeneration in epithelial cells damaged by the membrane-disrupting toxin, aerolysin (48). As lipids are sorted from the Golgi apparatus to the chlamydial inclusion (13, 15, 49), we therefore investigated whether C. trachomatis induces caspase-1 activation in epithelial cells via the assembly of an inflammasome. We demonstrated that C. trachomatis-induced caspase-1 activation is mediated by an inflammasome containing the NLR member, NLRP3. Several studies have demonstrated the involvement of T3S apparatus in inflammasome-mediated caspase-1 activation by different pathogens in macrophages and monocytes (50–56). Therefore, we further investigated the mechanism by which C. trachomatis triggers the formation of the NLRP3 inflammasome. Our results showed that metabolically active chlamydiae, relying on their T3S apparatus, cause K⁺ efflux, which in turn leads to formation of reactive oxygen species (ROS) and ultimately NLRP3-dependent caspase-1 activation. Epithelial cells do not typically secrete large amounts of IL-1β; instead, caspase-1 activation in cervical epithelial cells contributes to development of the chlamydial inclusion.     

Semaphorin3A Signaling Mediated by Fyn-dependent Tyrosine Phosphorylation of Collapsin Response Mediator Protein 2 at Tyrosine 32

Collapsin response mediator protein 2 (CRMP2) is an intracellular protein that mediates signaling of Semaphorin3A (Sema3A), a repulsive axon guidance molecule. Fyn, a Src-type tyrosine kinase, is involved in the Sema3A signaling. However, the relationship between CRMP2 and Fyn in this signaling pathway is still unknown. In our research, we demonstrated that Fyn phosphorylated CRMP2 at Tyr³² residues in HEK293T cells. Immunohistochemical analysis using a phospho-specific antibody at Tyr³² of CRMP showed that Tyr³²-phosphorylated CRMP was abundant in the nervous system, including dorsal root ganglion neurons, the molecular and Purkinje cell layer of adult cerebellum, and hippocampal fimbria. Overexpression of a nonphosphorylated mutant (Tyr³² to Phe³²) of CRMP2 in dorsal root ganglion neurons interfered with Sema3A-induced growth cone collapse response. These results suggest that Fyn-dependent phosphorylation of CRMP2 at Tyr³² is involved in Sema3A signaling.Collapsin response mediator proteins (CRMPs)⁴ have been identified as intracellular proteins that mediate Semaphorin3A (Sema3A) signaling in the nervous system (1). CRMP2 is one of the five members of the CRMP family. CRMPs also mediate signal transduction of NT3, Ephrin, and Reelin (2–4). CRMPs interact with several intracellular molecules, including tubulin, Numb, kinesin1, and Sra1 (5–8). CRMPs are involved in axon guidance, axonal elongation, cell migration, synapse maturation, and the generation of neuronal polarity (1, 2, 4, 5).CRMP family proteins are known to be the major phosphoproteins in the developing brain (1, 9). CRMP2 is phosphorylated by several Ser/Thr kinases, such as Rho kinase, cyclin-dependent kinase 5 (Cdk5), and glycogen synthase kinase 3β (GSK3β) (2, 10–13). The phosphorylation sites of CRMP2 by these kinases are clustered in the C terminus and have already been identified. Rho kinase phosphorylates CRMP2 at Thr⁵⁵⁵ (10). Cdk5 phosphorylates CRMP2 at Ser⁵²², and this phosphorylation is essential for sequential phosphorylations by GSK3β at Ser⁵¹⁸, Thr⁵¹⁴, and Thr⁵⁰⁹ (2, 11–13). These phosphorylations disrupt the interaction of CRMP2 with tubulin or Numb (2, 3, 13). The sequential phosphorylation of CRMP2 by Cdk5 and GSK3β is an essential step in Sema3A signaling (11, 13). Furthermore, the neurofibrillary tangles in the brains of people with Alzheimer disease contain hyperphosphorylated CRMP2 at Thr⁵⁰⁹, Ser⁵¹⁸, and Ser⁵²² (14, 15).CRMPs are also substrates of several tyrosine kinases. The phosphorylation of CRMP2 by Fes/Fps and Fer has been shown to be involved in Sema3A signaling (16, 17). Phosphorylation of CRMP2 at Tyr⁴⁷⁹ by a Src family tyrosine kinase Yes regulates CXCL12-induced T lymphocyte migration (18). We reported previously that Fyn is involved in Sema3A signaling (19). Fyn associates with PlexinA2, one of the components of the Sema3A receptor complex. Fyn also activates Cdk5 through the phosphorylation at Tyr¹⁵ of Cdk5 (19). In dorsal root ganglion (DRG) neurons from fyn-deficient mice, Sema3A-induced growth cone collapse response is attenuated compared with control mice (19). Furthermore, we recently found that Fyn phosphorylates CRMP1 and that this phosphorylation is involved in Reelin signaling (4). Although it has been shown that CRMP2 is involved in Sema3A signaling (1, 11, 13), the relationship between Fyn and CRMP2 in Sema3A signaling and the tyrosine phosphorylation site(s) of CRMPs remain unknown.Here, we show that Fyn phosphorylates CRMP2 at Tyr³². Using a phospho-specific antibody against Tyr³², we determined that the residue is phosphorylated in vivo. A nonphosphorylated mutant CRMP2Y32F inhibits Sema3A-induced growth cone collapse. These results indicate that tyrosine phosphorylation by Fyn at Tyr³² is involved in Sema3A signaling.