Structural Basis of Cyclic Nucleotide Selectivity in cGMP-dependent Protein Kinase II

Membrane-bound cGMP-dependent protein kinase (PKG) II is a key regulator of bone growth, renin secretion, and memory formation. Despite its crucial physiological roles, little is known about its cyclic nucleotide selectivity mechanism due to a lack of structural information. Here, we find that the C-terminal cyclic nucleotide binding (CNB-B) domain of PKG II binds cGMP with higher affinity and selectivity when compared with its N-terminal CNB (CNB-A) domain. To understand the structural basis of cGMP selectivity, we solved co-crystal structures of the CNB domains with cyclic nucleotides. Our structures combined with mutagenesis demonstrate that the guanine-specific contacts at Asp-412 and Arg-415 of the αC-helix of CNB-B are crucial for cGMP selectivity and activation of PKG II. Structural comparison with the cGMP selective CNB domains of human PKG I and Plasmodium falciparum PKG (PfPKG) shows different contacts with the guanine moiety, revealing a unique cGMP selectivity mechanism for PKG II.     

Protein Kinase D Mediates Mitogenic Signaling by Gq-coupled Receptors through Protein Kinase C-independent Regulation of Activation Loop Ser744 and Ser748 Phosphorylation

Rapid protein kinase D (PKD) activation and phosphorylation via protein kinase C (PKC) have been extensively documented in many cell types cells stimulated by multiple stimuli. In contrast, little is known about the role and mechanism(s) of a recently identified sustained phase of PKD activation in response to G protein-coupled receptor agonists. To elucidate the role of biphasic PKD activation, we used Swiss 3T3 cells because PKD expression in these cells potently enhanced duration of ERK activation and DNA synthesis in response to G_q-coupled receptor agonists. Cell treatment with the preferential PKC inhibitors GF109203X or Gö6983 profoundly inhibited PKD activation induced by bombesin stimulation for <15 min but did not prevent PKD catalytic activation induced by bombesin stimulation for longer times (>60 min). The existence of sequential PKC-dependent and PKC-independent PKD activation was demonstrated in 3T3 cells stimulated with various concentrations of bombesin (0.3–10 nm) or with vasopressin, a different G_q-coupled receptor agonist. To gain insight into the mechanisms involved, we determined the phosphorylation state of the activation loop residues Ser⁷⁴⁴ and Ser⁷⁴⁸. Transphosphorylation targeted Ser⁷⁴⁴, whereas autophosphorylation was the predominant mechanism for Ser⁷⁴⁸ in cells stimulated with G_q-coupled receptor agonists. We next determined which phase of PKD activation is responsible for promoting enhanced ERK activation and DNA synthesis in response to G_q-coupled receptor agonists. We show, for the first time, that the PKC-independent phase of PKD activation mediates prolonged ERK signaling and progression to DNA synthesis in response to bombesin or vasopressin through a pathway that requires epidermal growth factor receptor-tyrosine kinase activity. Thus, our results identify a novel mechanism of G_q-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.The understanding of the mechanisms that control cell proliferation requires the identification of the molecular pathways that govern the transition of quiescent cells into the S phase of the cell cycle. In this context the activation and phosphorylation of protein kinase D (PKD),⁴ the founding member of a new protein kinase family within the Ca²⁺/calmodulin-dependent protein kinase (CAMK) group and separate from the previously identified PKCs (for review, see Ref. 1), are attracting intense attention. In unstimulated cells, PKD is in a state of low catalytic (kinase) activity maintained by autoinhibition mediated by the N-terminal domain, a region containing a repeat of cysteinerich zinc finger-like motifs and a pleckstrin homology (PH) domain (1–4). Physiological activation of PKD within cells occurs via a phosphorylation-dependent mechanism first identified in our laboratory (5–7). In response to cellular stimuli (1), including phorbol esters, growth factors (e.g. PDGF), and G protein-coupled receptor (GPCR) agonists (6, 8–16) that signal through G_q, G₁₂, G_i, and Rho (11, 15–19), PKD is converted into a form with high catalytic activity, as shown by in vitro kinase assays performed in the absence of lipid co-activators (5, 20).During these studies multiple lines of evidence indicated that PKC activity is necessary for rapid PKD activation within intact cells. For example, rapid PKD activation was selectively and potently blocked by cell treatment with preferential PKC inhibitors (e.g. GF109203X or Gö6983) that do not directly inhibit PKD catalytic activity (5, 20), implying that PKD activation in intact cells is mediated directly or indirectly through PKCs. Many reports demonstrated the operation of a rapid PKC/PKD signaling cascade induced by multiple GPCR agonists and other receptor ligands in a range of cell types (for review, see Ref. 1). Our previous studies identified Ser⁷⁴⁴ and Ser⁷⁴⁸ in the PKD activation loop (also referred as activation segment or T-loop) as phosphorylation sites critical for PKC-mediated PKD activation (1, 4, 7, 17, 21). Collectively, these findings demonstrated the existence of a rapidly activated PKC-PKD protein kinase cascade(s). In a recent study we found that the rapid PKC-dependent PKD activation was followed by a late, PKC-independent phase of catalytic activation and phosphorylation induced by stimulation of the bombesin G_q-coupled receptor ectopically expressed in COS-7 cells (22). This study raised the possibility that PKD mediates rapid biological responses downstream of PKCs, whereas, in striking contrast, PKD could mediate long term responses through PKC-independent pathways. Despite its potential importance for defining the role of PKC and PKD in signal transduction, this hypothesis has not been tested in any cell type.Accumulating evidence demonstrates that PKD plays an important role in several cellular processes and activities, including signal transduction (14, 23–25), chromatin organization (26), Golgi function (27, 28), gene expression (29–31), immune regulation (26), and cell survival, adhesion, motility, differentiation, DNA synthesis, and proliferation (for review, see Ref. 1). In Swiss 3T3 fibroblasts, a cell line used extensively as a model system to elucidate mechanisms of mitogenic signaling (32–34), PKD expression potently enhances ERK activation, DNA synthesis, and cell proliferation induced by G_q-coupled receptor agonists (8, 14). Here, we used this model system to elucidate the role and mechanism(s) of biphasic PKD activation. First, we show that the G_q-coupled receptor agonists bombesin and vasopressin, in contrast to phorbol esters, specifically induce PKD activation through early PKC-dependent and late PKC-independent mechanisms in Swiss 3T3 cells. Subsequently, we demonstrate for the first time that the PKC-independent phase of PKD activation is responsible for promoting ERK signaling and progression to DNA synthesis through an epidermal growth factor receptor (EGFR)-dependent pathway. Thus, our results identify a novel mechanism of G_q-coupled receptor-induced mitogenesis mediated by sustained PKD activation through a PKC-independent pathway.     

Role of Ca2+/Calmodulin-dependent Protein Kinase II in Drosophila Photoreceptors

Ca²⁺ modulates the visual response in both vertebrates and invertebrates. In Drosophila photoreceptors, an increase of cytoplasmic Ca²⁺ mimics light adaptation. Little is known regarding the mechanism, however. We explored the role of the sole Drosophila Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) to mediate light adaptation. CaMKII has been implicated in the phosphorylation of arrestin 2 (Arr2). However, the functional significance of Arr2 phosphorylation remains debatable. We identified retinal CaMKII by anti-CaMKII antibodies and by its Ca²⁺-dependent autophosphorylation. Moreover, we show that phosphorylation of CaMKII is greatly enhanced by okadaic acid, and indeed, purified PP2A catalyzes the dephosphorylation of CaMKII. Significantly, we demonstrate that anti-CaMKII antibodies co-immunoprecipitate, and CaMKII fusion proteins pull down the catalytic subunit of PP2A from fly extracts, indicating that PP2A interacts with CaMKII to form a protein complex. To investigate the function of CaMKII in photoreceptors, we show that suppression of CaMKII in transgenic flies affects light adaptation and increases prolonged depolarizing afterpotential amplitude, whereas a reduced PP2A activity brings about reduced prolonged depolarizing afterpotential amplitude. Taken together, we conclude that CaMKII is involved in the negative regulation of the visual response affecting light adaptation, possibly by catalyzing phosphorylation of Arr2. Moreover, the CaMKII activity appears tightly regulated by the co-localized PP2A.Visual transduction is the process that converts the signal of light (photons) into a change of membrane potential in photoreceptors (see Ref. 1 for review). Visual signaling is initiated upon the activation of rhodopsins by light: light switches on rhodopsin to generate metarhodopsin, which activates the heterotrimeric G_q in Drosophila (2). Subsequently, the GTP-bound Gα_q subunit activates phospholipase Cβ4 encoded by the norpA (no receptor potential A) gene (3). Phospholipase Cβ4 catalyzes the breakdown of phosphoinositol 4,5-bisphosphate to generate diacylglycerol, which or its metabolite has been implicated in gating the transient receptor potential (TRP)² and TRP-like channels (4, 5). TRP is the major Ca²⁺ channel that mediates the light-dependent depolarization response leading to an increase of cytosolic Ca²⁺ in photoreceptors. The rise of intracellular Ca²⁺ modulates several aspects of the visual response including activation, deactivation, and light adaptation (6). For example, Ca²⁺ together with diacylglycerol activates a classical protein kinase C, eye-PKC, which is critical for the negative regulation of visual signaling by modulating deactivation and light adaptation (7–11).Light adaptation is the process by which photoreceptors adjust the visual sensitivity in response to ambient background light by down-regulating rhodopsin-mediated signaling. Light adaptation can be arbitrarily subdivided into long term and short term adaptation and may involve multiple regulations to reduce the efficiency of rhodopsin, G protein, or cation channels. For example, translocation of both G_q (12, 13) and TRP-like channels (14, 15) out of the visual organelle may contribute to long term adaptation in Drosophila. In contrast, short term adaptation may be orchestrated by modulating the activity of signaling proteins by protein kinases. Hardie and co-workers (16) demonstrated that an increase of cytoplasmic [Ca²⁺] mimicked light adaptation, leading to inhibition of the light-induced current. These authors also showed that light adaptation is independent of eye-PKC. Thus the effect of cytoplasmic Ca²⁺ to control light adaptation is likely mediated via calmodulin and CaMKII. The contribution of CaMKII to light adaptation has not been explored.CaMKII is a multimeric Ca²⁺/calmodulin-dependent protein kinase that modulates diverse signaling processes (17). Drosophila contains one CaMKII gene (18) that gives rise to at least four protein isoforms (19). These CaMKII isoforms share over 85% sequence identities with the α isoform of vertebrate CaMKII. For insights into the in vivo physiological role of CaMKII, Griffith et al. (20) generated transgenic flies (ala) expressing an inhibitory domain of the rat CaMKII under the control of a heat shock promoter, hsp70. They demonstrated that, upon heat shock treatment, the overexpression of the inhibitory peptide resulted in a suppression of the endogenous CaMKII activity in the transgenic flies (20). It has been shown that inhibition of CaMKII affects learning and memory (20) and neuronal functions (21–24). In photoreceptors, CaMKII has been implicated in the phosphorylation of the major visual arrestin, Arr2 (25, 26). However, how phosphorylation of Arr2 by CaMKII modifies the visual signaling remains to be elucidated.Here we report the biochemical and electrophysiological analyses of CaMKII in Drosophila retina. We demonstrate that suppression of CaMKII in ala¹ transgenic flies leads to a phenotype indicative of defective light adaptation. The ala¹ flies also display greater visual response, suggesting a defect in Arr2. These results support the notion that CaMKII plays a role in the negative regulation of the visual response. Our biochemical analyses demonstrate that dephosphorylation of CaMKII is mediated by protein phosphatase 2A (PP2A). Importantly, we show that PP2A interacts with CaMKII, indicating that CaMKII forms a stable protein complex with PP2A to ensure a tight regulation of the kinase activity. Thus a partial loss of function in PP2A would elevate the CaMKII activity. Indeed, we show that mts heterozygotes display reduced prolonged depolarizing potential (PDA) amplitude. This PDA phenotype strongly suggests that Arr2 becomes more effective to terminate the visual signaling in mts flies. Together, our findings indicate that the ability of Arr2 to terminate metarhodopsin is increased upon phosphorylation by CaMKII, and the retinal CaMKII activity is regulated by PP2A.     

Protein Kinase Cδ Mediates Neurogenic but Not Mitogenic Activation of Mitogen-Activated Protein Kinase in Neuronal Cells

In several neuronal cell systems, fibroblast-derived growth factor (FGF) and nerve growth factor (NGF) act as neurogenic agents, whereas epidermal growth factor (EGF) acts as a mitogen. The mechanisms responsible for these different cellular fates are unclear. We report here that although FGF, NGF, and EGF all activate mitogen-activated protein (MAP) kinase (extracellular signal-related kinase [ERK]) in rat hippocampal (H19-7) and pheochromocytoma (PC12) cells, the activation of ERK by the neurogenic agents FGF and NGF is dependent upon protein kinase Cdelta (PKCdelta), whereas ERK activation in response to the mitogenic EGF is independent of PKCdelta. Antisense PKCdelta oligonucleotides or the PKCdelta-specific inhibitor rottlerin inhibited FGF- and NGF-induced, but not EGF-induced, ERK activation. In contrast, EGF-induced ERK activation was inhibited by the phosphatidylinositol-3-kinase inhibitor wortmannin, which had no effect upon FGF-induced ERK activation. Rottlerin also inhibited the activation of MAP kinase kinase (MEK) in response to activated Raf, but had no effect upon c-Raf activity or ERK activation by activated MEK. These results indicate that PKCdelta functions either downstream from or in parallel with c-Raf, but upstream of MEK. Inhibition of PKCdelta also blocked neurite outgrowth induced by FGF and NGF in PC12 cells and by activated Raf in H19-7 cells, indicating a role for PKCdelta in the neurogenic effects of FGF, NGF, and Raf. Interestingly, the PKCdelta requirement is apparently cell type specific, since FGF-induced ERK activation was independent of PKCdelta in NIH 3T3 murine fibroblasts, in which FGF is a mitogen. These data demonstrate that PKCdelta contributes to growth factor specificity and response in neuronal cells and may also promote cell-type-specific differences in growth factor signaling.     

Protein Kinase C-Epsilon Mediates Phorbol Ester-Induced Phosphorylation of Connexin-43

We have used adenoviral vectors to express dominant negative variants of protein kinase C epsilon (PKC?) or mitogen kinase kinase 1 (MKK1) to investigate their involvement in phorbol ester-induced connexin-43 (Cx43) phosphorylation in cardiomyocytes. Stimulation of cardiomyocytes with phorbol 12-myristate 13-acetate (PMA) increased the fraction of the slower migrating (≥45 kDa) and more extensively phosphorylated Cx43 species. Expression of dominant negative MKKI did not prevent the effect of PMA on Cx43 phosphorylation. Selective inhibition of PKC? significantly decreased baseline levels of Cx43 phosphorylation and the PMA-induced accumulation of ≥45 kDa Cx43. Thus, production of the more extensively phosphorylated species of Cx43 in cardiomyocytes by PMA requires activation of PKC?.     

Role of Plasmodium berghei cGMP-dependent Protein Kinase in Late Liver Stage Development

The liver is the first organ infected by Plasmodium sporozoites during malaria infection. In the infected hepatocytes, sporozoites undergo a complex developmental program to eventually generate hepatic merozoites that are released into the bloodstream in membrane-bound vesicles termed merosomes. Parasites blocked at an early developmental stage inside hepatocytes elicit a protective host immune response, making them attractive targets in the effort to develop a pre-erythrocytic stage vaccine. Here, we generated parasites blocked at a late developmental stage inside hepatocytes by conditionally disrupting the Plasmodium berghei cGMP-dependent protein kinase in sporozoites. Mutant sporozoites are able to invade hepatocytes and undergo intracellular development. However, they remain blocked as late liver stages that do not release merosomes into the medium. These late arrested liver stages induce protection in immunized animals. This suggests that, similar to the well studied early liver stages, late stage liver stages too can confer protection from sporozoite challenge.     

Crystal Structure of the cGMP-dependent Protein Kinase II Leucine Zipper and Rab11b Protein Complex Reveals Molecular Details of G-kinase-specific Interactions

cGMP-dependent protein kinase (PKG)-interacting proteins (GKIPs) mediate cellular targeting of PKG isoforms by interacting with their leucine zipper (LZ) domains. These interactions prevent aberrant signaling cross-talk between different PKG isotypes. To gain detailed insight into isotype-specific GKIP recognition by PKG, we analyzed the type II PKG leucine zipper domain and found that residues 40–83 dimerized and specifically interacted with Rab11b. Next, we determined a crystal structure of the PKG II LZ-Rab11b complex. The PKG II LZ domain presents a mostly nonpolar surface onto which Rab11b docks, through van der Waals interactions. Contact surfaces in Rab11b are found in switch I and II, interswitch, and the β1/N-terminal regions. This binding surface dramatically differs from that seen in the Rab11 family of interacting protein complex structures. Structural comparison with PKG Iα and Iβ LZs combined with mutagenic analysis reveals that GKIP recognition is mediated through surface charge interactions.     

Phospholipase D1 Mediates AMP-Activated Protein Kinase Signaling for Glucose Uptake

Background

Glucose homeostasis is maintained by a balance between hepatic glucose production and peripheral glucose utilization. In skeletal muscle cells, glucose utilization is primarily regulated by glucose uptake. Deprivation of cellular energy induces the activation of regulatory proteins and thus glucose uptake. AMP-activated protein kinase (AMPK) is known to play a significant role in the regulation of energy balances. However, the mechanisms related to the AMPK-mediated control of glucose uptake have yet to be elucidated.

Methodology/Principal Findings

Here, we found that AMPK-induced phospholipase D1 (PLD1) activation is required for ¹⁴C-glucose uptake in muscle cells under glucose deprivation conditions. PLD1 activity rather than PLD2 activity is significantly enhanced by glucose deprivation. AMPK-wild type (WT) stimulates PLD activity, while AMPK-dominant negative (DN) inhibits it. AMPK regulates PLD1 activity through phosphorylation of the Ser-505 and this phosphorylation is increased by the presence of AMP. Furthermore, PLD1-S505Q, a phosphorylation-deficient mutant, shows no changes in activity in response to glucose deprivation and does not show a significant increase in ¹⁴C-glucose uptake when compared to PLD1-WT. Taken together, these results suggest that phosphorylation of PLD1 is important for the regulation of ¹⁴C-glucose uptake. In addition, extracellular signal-regulated kinase (ERK) is stimulated by AMPK-induced PLD1 activation through the formation of phosphatidic acid (PA), which is a product of PLD. An ERK pharmacological inhibitor, PD98059, and the PLD inhibitor, 1-BtOH, both attenuate ¹⁴C-glucose uptake in muscle cells. Finally, the extracellular stresses caused by glucose deprivation or aminoimidazole carboxamide ribonucleotide (AICAR; AMPK activator) regulate ¹⁴C-glucose uptake and cell surface glucose transport (GLUT) 4 through ERK stimulation by AMPK-mediated PLD1 activation.

Conclusions/Significance

These results suggest that AMPK-mediated PLD1 activation is required for ¹⁴C-glucose uptake through ERK stimulation. We propose that the AMPK-mediated PLD1 pathway may provide crucial clues to understanding the mechanisms involved in glucose uptake.     

GroEL Mediates Folding of <Emphasis Type="Italic">Bacillus anthracis</Emphasis> Serine/Threonine Protein Kinase,PrkC

Bacillus anthracis causes anthrax in human and animals. Both, signaling system such as two component system and endogenous chaperone system such as GroEL–GroES help bacteria to cope with the environmental challenges. Such molecular chaperones are the stress induced proteins that help bacteria to override unfavorable conditions by their moonlighting functions. Previous reports showed that PrkC and PrpC, the Ser/Thr kinase–phosphatase pair in B. anthracis, control phosphorylation of GroEL and regulate biofilm formation. In this study, we show that GroEL is involved in the folding of PrkC to active form. The proteins (GroEL, PrkC and PrpC) were expressed and purified by affinity chromatography. Purified GroEL was used for refolding of denatured PrkC and PrpC and observed that GroEL refolds PrkC but not PrpC as measured by their enzymatic activity. We also observed that purification of GroEL with six histidine tag using Cobalt-Agarose resin yielded superior quality GroEL protein with negligible contamination of non-specific proteins. Thus, cobalt resin can be a better choice for purification of many histidine tagged proteins, where Ni-NTA does not work very well.     

Oxidative Stress Induced Mitochondrial Protein Kinase A Mediates Cytochrome C Oxidase Dysfunction

Previously we showed that Protein kinase A (PKA) activated in hypoxia and myocardial ischemia/reperfusion mediates phosphorylation of subunits I, IVi1 and Vb of cytochrome c oxidase. However, the mechanism of activation of the kinase under hypoxia remains unclear. It is also unclear if hypoxic stress activated PKA is different from the cAMP dependent mitochondrial PKA activity reported under normal physiological conditions. In this study using RAW 264.7 macrophages and in vitro perfused mouse heart system we investigated the nature of PKA activated under hypoxia. Limited protease treatment and digitonin fractionation of intact mitochondria suggests that higher mitochondrial PKA activity under hypoxia is mainly due to increased sequestration of PKA Catalytic α (PKAα) subunit in the mitochondrial matrix compartment. The increase in PKA activity is independent of mitochondrial cAMP and is not inhibited by adenylate cyclase inhibitor, KH7. Instead, activation of hypoxia-induced PKA is dependent on reactive oxygen species (ROS). H89, an inhibitor of PKA activity and the antioxidant Mito-CP prevented loss of CcO activity in macrophages under hypoxia and in mouse heart under ischemia/reperfusion injury. Substitution of wild type subunit Vb of CcO with phosphorylation resistant S40A mutant subunit attenuated the loss of CcO activity and reduced ROS production. These results provide a compelling evidence for hypoxia induced phosphorylation as a signal for CcO dysfunction. The results also describe a novel mechanism of mitochondrial PKA activation which is independent of mitochondrial cAMP, but responsive to ROS.     

Sustained Protein Kinase D Activation Mediates Respiratory Syncytial Virus-Induced Airway Barrier Disruption

Understanding the regulation of airway epithelial barrier function is a new frontier in asthma and respiratory viral infections. Despite recent progress, little is known about how respiratory syncytial virus (RSV) acts at mucosal sites, and very little is known about its ability to influence airway epithelial barrier function. Here, we studied the effect of RSV infection on the airway epithelial barrier using model epithelia. 16HBE14o- bronchial epithelial cells were grown on Transwell inserts and infected with RSV strain A2. We analyzed (i) epithelial apical junction complex (AJC) function, measuring transepithelial electrical resistance (TEER) and permeability to fluorescein isothiocyanate (FITC)-conjugated dextran, and (ii) AJC structure using immunofluorescent staining. Cells were pretreated or not with protein kinase D (PKD) inhibitors. UV-irradiated RSV served as a negative control. RSV infection led to a significant reduction in TEER and increase in permeability. Additionally it caused disruption of the AJC and remodeling of the apical actin cytoskeleton. Pretreatment with two structurally unrelated PKD inhibitors markedly attenuated RSV-induced effects. RSV induced phosphorylation of the actin binding protein cortactin in a PKD-dependent manner. UV-inactivated RSV had no effect on AJC function or structure. Our results suggest that RSV-induced airway epithelial barrier disruption involves PKD-dependent actin cytoskeletal remodeling, possibly dependent on cortactin activation. Defining the mechanisms by which RSV disrupts epithelial structure and function should enhance our understanding of the association between respiratory viral infections, airway inflammation, and allergen sensitization. Impaired barrier function may open a potential new therapeutic target for RSV-mediated lung diseases.     

The Ras GTPase-activating-like Protein IQGAP1 Mediates Nrf2 Protein Activation via the Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase (ERK) Kinase (MEK)-ERK Pathway

Nrf2 plays a critical role in the regulation of cellular oxidative stress. MEK-ERK activation has been shown to be one of the major pathways resulting in the activation of Nrf2 and induction of Nrf2 downstream targets, including phase II detoxifying/antioxidant genes in response to oxidative stress and xenobiotics. In this study, IQGAP1 (IQ motif-containing GTPase-activating protein 1), a new Nrf2 interaction partner that we have published previously, was found to modulate MEK-ERK-mediated Nrf2 activation and induction of phase II detoxifying/antioxidant genes. Nrf2 binds directly to the IQ domain (amino acids 699–905) of IQGAP1. Knockdown of IQGAP1 significantly attenuated phenethyl isothiocyanate- or MEK-mediated activation of the MEK-ERK-Nrf2 pathway. Knockdown of IQGAP1 also attenuated MEK-mediated increased stability of Nrf2, which in turn was associated with a decrease in the nuclear translocation of Nrf2 and a decrease in the expression of phase II detoxifying/antioxidant genes. In the aggregate, these results suggest that IQGAP1 may play an important role in the MEK-ERK-Nrf2 signaling pathway.     

Neuregulin Mediates F-actin-driven Cell Migration through Inhibition of Protein Kinase D1 via Rac1 Protein

Neuregulin (NRG; heregulin) is overexpressed in ∼30% of breast cancers and mediates various processes involved in tumor progression, including tumor cell migration and invasion. Here, we show that NRG mediates its effects on tumor cell migration via PKD1. Downstream of RhoA, PKD1 can prevent directed cell migration through phosphorylation of its substrate SSH1L. NRG exerts its inhibitory effects on PKD1 through Rac1/NADPH oxidase, leading to decreased PKD1 activation loop phosphorylation and decreased activity toward SSH1L. The consequence of PKD1 inhibition by NRG is decreased binding of 14-3-3 to SSH1L, localization of SSH1L to F-actin at the leading edge, and increased cofilin activity, resulting in increased reorganization of the actin cytoskeleton and cell motility. Our data provide a mechanism through which the Rho GTPase Rac1 cross-talks with PKD1 signaling pathways to facilitate directed cell migration.     

Spatial Distribution of Protein Kinase A Activity during Cell Migration Is Mediated by A-kinase Anchoring Protein AKAP Lbc

Protein kinase A (PKA) has been suggested to be spatially regulated in migrating cells due to its ability to control signaling events that are critical for polarized actin cytoskeletal dynamics. Here, using the fluorescence resonance energy transfer-based A-kinase activity reporter (AKAR1), we find that PKA activity gradients form with the strongest activity at the leading edge and are restricted to the basal surface in migrating cells. The existence of these gradients was confirmed using immunocytochemistry using phospho-PKA substrate antibodies. This observation holds true for carcinoma cells migrating randomly on laminin-1 or stimulated to migrate on collagen I with lysophosphatidic acid. Phosphodiesterase inhibition allows the formation of PKA activity gradients; however, these gradients are no longer polarized. PKA activity gradients are not detected when a non-phosphorylatable mutant of AKAR1 is used, if PKA activity is inhibited with H-89 or protein kinase inhibitor, or when PKA anchoring is perturbed. We further find that a specific A-kinase anchoring protein, AKAP-Lbc, is a major contributor to the formation of these gradients. In summary, our data show that PKA activity gradients are generated at the leading edge of migrating cells and provide additional insight into the mechanisms of PKA regulation of cell motility.Cell motility is controlled by a complex network of signals that are initiated by binding to the extracellular matrix. Understanding the biochemical mechanisms that control cell migration is necessary for better comprehension of processes like wound healing, embryonic development, and angiogenesis as well as cancer metastasis (1). PKA³ is an important regulator of cell signaling and various biological functions (2-4). Previous studies have shown that cell motility is delicately controlled by synthesis and breakdown of cAMP through its effects on PKA. PKA regulates key signaling events that are critical for actin cytoskeletal remodeling and cell polarization during migration, including control of the activation states of RhoA, Rac, cdc42, Pak, and c-Abl. For example, PKA is known to inhibit the activation of RhoA, whereas it is required for the activation of Rac1, two proteins that are spatially regulated during cell migration. Therefore, it has been suggested that PKA activity in migrating cells is spatially regulated (5-9). The mounting evidence for the formation of cAMP/PKA gradients and their influence over directed cell motility is compelling. To conclusively determine that PKA activity gradients exist, the visualization of these gradients in single cells is needed to determine the nature of gradients and the mechanisms governing how they are formed.The compartmental action of cAMP was suggested over three decades ago (10, 11) and has hence been shown to mediate the precise spatiotemporal control of its effectors (12-15). Tight control of cAMP levels is governed by the coordinated actions of cyclic nucleotide phosphodiesterases (PDEs) and adenylyl cyclases. Gradients of cAMP and, thus, PKA activity are expected to exist in a cell. This idea is based, most simplistically, on the fact that cAMP is generated by membrane-bound adenylyl cyclases and broken down by cytosolic PDEs; that is, the two arms of cAMP metabolism are spatially separated. Further compartmentalization of PKA activity also occurs as a result of the anchoring of PKA and cAMP-specific PDEs to A-kinase anchoring proteins (AKAPs), which has been demonstrated in a variety of cell types (16, 17). The anchoring of PKA occurs typically through the binding of the type II regulatory (RII) subunits to AKAPs where the relative levels of PDE activity and cAMP generated regulate the regional activity of PKA. PKA anchoring, in addition to cAMP synthesis and degradation, is believed to control spatial signaling of PKA (14, 15). Until recently, we have lacked both the model systems and technology to adequately study the possibility that cAMP/PKA activity gradients exist. We and others (5-8) have established that polarization and migration of cells are dependent on cAMP synthesis and breakdown. Here, we sought to demonstrate the existence of cAMP/PKA gradients in single migrating cells using the fluorescence resonance energy transfer (FRET)-based PKA biosensor A-kinase activity reporter (AKAR1) and determine how signaling components that regulate PKA activity, including cAMP synthesis, PDEs, and PKA anchoring, affect the formation of these gradients.     

Stress-Activated Protein Kinase/c-Jun N-Terminal Kinase Phosphorylates τ Protein

Abstract: A proportion of the neuronal microtubule-associated protein (MAP) τ is highly phosphorylated in foetal and adult brain, whereas the majority of τ in the neurofibrillary tangles of Alzheimer's patients is hyperphosphorylated; many of the phosphorylation sites are serines or threonines followed by prolines. Several kinases phosphorylate τ at such sites in vitro. We have now shown that purified recombinant stress-activated protein kinase/c-Jun N-terminal kinase, a proline-directed kinase of the MAP kinase extended family, phosphorylates recombinant τ in vitro on threonine and serine residues. Western blots using antibodies to phosphorylation-dependent τ epitopes demonstrated that phosphorylation occurs in both of the main phosphorylated regions of τ protein. Unlike glycogen synthase kinase-3, the c-Jun N-terminal kinase readily phosphorylates Thr²⁰⁵ and Ser⁴²², which are more highly phosphorylated in Alzheimer τ than in foetal or adult τ. Glycogen synthase kinase-3 may preferentially phosphorylate the sites found physiologically, in foetal and to a smaller extent in adult τ, whereas stress-activated/c-Jun N-terminal kinase and/or other members of the extended MAP kinase family may be responsible for pathological proline-directed phosphorylations. Inflammatory processes in Alzheimer brain might therefore contribute directly to the pathological formation of the hyperphosphorylated τ found in neurofibrillary tangles.     

Down-Regulation of Decapping Protein 2 Mediates Chronic Nicotine Exposure-Induced Locomotor Hyperactivity in Drosophila

Long-term tobacco use causes nicotine dependence via the regulation of a wide range of genes and is accompanied by various health problems. Studies in mammalian systems have revealed some key factors involved in the effects of nicotine, including nicotinic acetylcholine receptors (nAChRs), dopamine and other neurotransmitters. Nevertheless, the signaling pathways that link nicotine-induced molecular and behavioral modifications remain elusive. Utilizing a chronic nicotine administration paradigm, we found that adult male fruit flies exhibited locomotor hyperactivity after three consecutive days of nicotine exposure, while nicotine-naive flies did not. Strikingly, this chronic nicotine-induced locomotor hyperactivity (cNILH) was abolished in Decapping Protein 2 or 1 (Dcp2 or Dcp1) -deficient flies, while only Dcp2-deficient flies exhibited higher basal levels of locomotor activity than controls. These results indicate that Dcp2 plays a critical role in the response to chronic nicotine exposure. Moreover, the messenger RNA (mRNA) level of Dcp2 in the fly head was suppressed by chronic nicotine treatment, and up-regulation of Dcp2 expression in the nervous system blocked cNILH. These results indicate that down-regulation of Dcp2 mediates chronic nicotine-exposure-induced locomotor hyperactivity in Drosophila. The decapping proteins play a major role in mRNA degradation; however, their function in the nervous system has rarely been investigated. Our findings reveal a significant role for the mRNA decapping pathway in developing locomotor hyperactivity in response to chronic nicotine exposure and identify Dcp2 as a potential candidate for future research on nicotine dependence.