Phosphorylation of the Cool-1/β-Pix Protein Serves as a Regulatory Signal for the Migration and Invasive Activity of Src-transformed Cells

Previously we showed that Cool-1 (Cloned out of library-1)/β-Pix (Pak-interactive exchange factor) is phosphorylated at a specific tyrosine residue (Tyr-442) in a Src-dependent manner and serves as a dual function guanine nucleotide exchange factor (GEF)/signaling-effector for Cdc42 that is essential for transformation by Src. Here, we show that knocking-down Cool-1 or overexpressing a Cool-1 mutant that contains substitutions within its Dbl homology domain and is defective for GEF activity, inhibits Src-promoted cell migration. Similarly, the expression of a Cool-1 mutant containing a tyrosine to phenylalanine substitution at position 442, making it incapable of being phosphorylated in response to serum, epidermal growth factor (EGF), or Src, also causes a significant inhibition of the migration and invasive activity of cells expressing oncogenic Src. We further demonstrate that the phosphorylation of Cool-1 at Tyr-442 weakens its ability to bind to one of its primary interaction-partners, Cat-1 (Cool-associated tyrosine phosphosubstrate-1)/Git-1 (G protein-coupled receptor kinase-interactor-1), thus making Cat more accessible for binding to paxillin. This enables cells to alternate between states where they contain large numbers of focal complexes (i.e. conditions favoring Cool-1-Cat interactions) versus reduced numbers of focal complexes (conditions favoring Cat-paxillin interactions). Overall, these findings show that the phosphorylation-dephosphorylation cycle of Cool-1 at Tyr-442 can serve as a key regulatory signal for focal complex assembly-disassembly, and consequently, for the migration and invasive activity of Src-transformed cells.     

Ran GTPase guanine nucleotide exchange factor RCC1 is phosphorylated on serine 11 by cdc2 kinase in vitro

RCC1, a guanine nucleotide exchange factor for Ran GTPase, plays essential roles in the growth and viability of mammalian cells. Here, we examined the phosphorylation of specific serine and threonine residues of RCC1 in vivo and showed that RCC1 is indeed phosphorylated. Analysis by two-dimensional (2D) gel electrophoresis suggested that serine 11 (S11) of hamster RCC1 is phosphorylated in vivo. A point mutation of S11 of hamster RCC1 resulted in a decrease in the number of 2D gel spots, indicating a lack of phosphorylation at the mutant residue. S11 phosphorylation in vitro depended on cyclin B-cdc2 kinase. An RCC1 mutant in which all N-terminal serine and threonine residues were substituted with glutamate residues to mimic phosphorylation at these residues showed decreased binding to the karyopherin, KPNA4, compared with wild type RCC1. We conclude that RCC1 undergoes post-translational phosphorylation.     

The N-terminal coiled-coil domain of the cytohesin/ARNO family of guanine nucleotide exchange factors interacts with Gαq

Cytohesins are guanine-nucleotide exchange factors (GEF) for the Arf family of GTPases. One member of the Arf family, ARF6, plays an active role in the intracellular trafficking of G protein-coupled receptors. We have previously reported that Gαq signaling leads to the activation of ARF6, possibly through a direct interaction with cytohesin-2/ARNO. Here, we report that Gαq can directly interact with cytohesin-1, another Arf-GEF of the ARNO/cytohesin family. Cytohesin-1 preferentially associated with a constitutively active mutant of Gαq (Gαq-Q209L) compared to wild-type Gαq in HEK293 cells. Stimulation of TPβ, a Gαq-coupled receptor, to activate Gαq resulted in the promotion of a protein complex between Gαq and cytohesin-1. Confocal immunofluorescence microscopy revealed that wild-type Gαq and cytohesin-1 co-localized in intracellular compartments and at or near the plasma membrane. In contrast, expression of Gαq-Q209L induced a drastic increase in the localization of cytohesin-1 at the plasma membrane. Expression of a dominant-negative mutant of cytohesin-1 reduced by 40% the agonist-induced internalization of TPβ, a process that we previously demonstrated to be dependent on Gαq-mediated signaling and Arf6 activation. Using deletion mutants, we show that cytohesin-1 interacts with Gαq through its N-terminal coiled-coil domain. Cytohesin-1 and cytohesin-2/ARNO mutants lacking the coiled-coil domain were unable to relay Gαq-mediated activation of Arf6. This is the first report of an interaction between the coiled-coil domain of the cytohesin/ARNO family of Arf-GEFs and a member of the heterotrimeric G proteins.     

MEGF10 functions as a receptor for the uptake of amyloid-β

MEGF10 is predominantly expressed in the brain and known to function as a phagocytic receptor. Here, we provide evidence that MEGF10 is involved in the uptake of amyloid-β peptide (Aβ42) in the brain. Overexpression of MEGF10 dramatically increased Aβ42 uptake in Hela cells. Knockdown of endogenous MEGF10 expression significantly decreased Aβ42 uptake in N2A neuroblastoma cells. MEGF10-mediated Aβ uptake is mostly dependent on lipid raft endocytosis pathway. Furthermore, site-directed mutagenesis revealed that the conserved cytoplasmic NPxY and YxxØ motifs are crucial for MEGF10-mediated uptake of Aβ42 peptide. Thus, the identification of the MEGF10 as a functional receptor that mediates the uptake of amyloid-β peptide will help elucidate the molecular mechanisms of amlyoid-β clearance in Alzheimer’s disease.

Structured summary

MINT-7993537: ctxB (uniprotkb:P01556) and Abeta (uniprotkb:P05067) colocalize (MI:0403) by fluorescence microscopy (MI:0416)     

β-Arrestin1 mediates the endocytosis and functions of macrophage migration inhibitory factor

Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine, regulating inflammatory and immune responses. MIF binds to cell surface receptor CD74, resulting in both rapid and sustained ERK activation. It was reported that MIF-induced rapid ERK activation requires its co-receptor CD44. But the exact mechanism underlying sustained ERK activation is not well understood. In the current study, we described a detailed mechanism of MIF mediated sustained ERK activation. We found that β-arrestin1, a scaffold protein involved in the activation of the MAPK cascade, interacts with CD74 upon MIF stimulation, resulting in CD74-mediated MIF endocytosis in a chlorpromazine (CPZ)-sensitive manner. β-arrestin1 is also involved in endocytotic MIF signaling, leading to sustained ERK activation. Therefore β-arrestin1 plays a central role in coupling MIF endocytosis to sustained ERK activation.     

Oct-1 functions as a transactivator in the hormonal induction of β-casein gene expression

An adverse environmental experience of the growing fetus leads to permanent changes in the structure and contractile function of the heart; however, the mechanisms are incompletely understood. To examine if a maternal low protein (LP) diet can modulate the gene and protein expression of the Ca²⁺-cycling proteins in the neonatal heart, we employed a rat model in which pregnant dams were fed diets containing either 180 (normal) or 90 g (low) casein/kg diet for 2 weeks before mating and throughout pregnancy. A significant reduction in the L-type Ca²⁺-channel mRNA level in the LP group was detected at 1, 7, and 14 days of age. Although ryanodine receptor (RyR) mRNA levels progressively declined in the aging heart in both groups, the RyR mRNA levels were consistently higher in the LP group. A reduction in RyR protein content was seen only in the hearts of the LP group at 7 days of age. The Na⁺-Ca²⁺-exchanger (NCX) mRNA level was also markedly increased at all ages. Although an increase in sarco(endo)plasmic reticulum ATPase 2a (SERCA) 2a mRNA was only detected in the LP group at 7 days of age, corresponding protein level was depressed. On the other hand, an initial decrease (at 1 day of age) followed by an increase (at 14 and 28 days of age) in phospholamban (PLB) mRNA levels was detected. Although PLB protein level was also depressed at 1 day of age in the LP group, a marked increase was seen at 7 days of age. Moreover, the ratio of serine 16 and threonine 17 phosphorylated PLB to non-phosphorylated PLB was reduced at 7 days of age in the hearts of offspring of the LP group. These data suggest that maternal LP diet can induce alterations in the gene expression and protein levels of the Ca²⁺-cycling proteins in the neonatal heart.     

The nucleotide exchange factor Ric-8A is a chaperone for the conformationally dynamic nucleotide-free state of Gαi1

Heterotrimeric G protein α subunits are activated upon exchange of GDP for GTP at the nucleotide binding site of Gα, catalyzed by guanine nucleotide exchange factors (GEFs). In addition to transmembrane G protein-coupled receptors (GPCRs), which act on G protein heterotrimers, members of the family cytosolic proteins typified by mammalian Ric-8A are GEFs for Gi/q/12/13-class Gα subunits. Ric-8A binds to Gα?GDP, resulting in the release of GDP. The Ric-8A complex with nucleotide-free Gαi1 is stable, but dissociates upon binding of GTP to Gαi1. To gain insight into the mechanism of Ric-8A-catalyzed GDP release from Gαi1, experiments were conducted to characterize the physical state of nucleotide-free Gαi1 (hereafter referred to as Gαi1[ ]) in solution, both as a monomeric species, and in the complex with Ric-8A. We found that Ric-8A-bound, nucleotide-free Gαi1 is more accessible to trypsinolysis than Gαi1?GDP, but less so than Gαi1[ ] alone. The TROSY-HSQC spectrum of [(15)N]Gαi1[ ] bound to Ric-8A shows considerable loss of peak intensity relative to that of [(15)N]Gαi1?GDP. Hydrogen-deuterium exchange in Gαi1[ ] bound to Ric-8A is 1.5-fold more extensive than in Gαi1?GDP. Differential scanning calorimetry shows that both Ric-8A and Gαi1?GDP undergo cooperative, irreversible unfolding transitions at 47° and 52°, respectively, while nucleotide-free Gαi1 shows a broad, weak transition near 35°. The unfolding transition for Ric-8A:Gαi1[ ] is complex, with a broad transition that peaks at 50°, suggesting that both Ric-8A and Gαi1[ ] are stabilized within the complex, relative to their respective free states. The C-terminus of Gαi1 is shown to be a critical binding element for Ric-8A, as is also the case for GPCRs, suggesting that the two types of GEF might promote nucleotide exchange by similar mechanisms, by acting as chaperones for the unstable and dynamic nucleotide-free state of Gα.     

αvβ8 integrin interacts with RhoGDI1 to regulate Rac1 and Cdc42 activation and drive glioblastoma cell invasion

The malignant brain cancer glioblastoma multiforme (GBM) displays invasive growth behaviors that are regulated by extracellular cues within the neural microenvironment. The adhesion and signaling pathways that drive GBM cell invasion remain largely uncharacterized. Here we use human GBM cell lines, primary patient samples, and preclinical mouse models to demonstrate that integrin αvβ8 is a major driver of GBM cell invasion. β8 integrin is overexpressed in many human GBM cells, with higher integrin expression correlating with increased invasion and diminished patient survival. Silencing β8 integrin in human GBM cells leads to impaired tumor cell invasion due to hyperactivation of the Rho GTPases Rac1 and Cdc42. β8 integrin coimmunoprecipitates with Rho-GDP dissociation inhibitor 1 (RhoGDI1), an intracellular signaling effector that sequesters Rho GTPases in their inactive GDP-bound states. Silencing RhoGDI1 expression or uncoupling αvβ8 integrin–RhoGDI1 protein interactions blocks GBM cell invasion due to Rho GTPase hyperactivation. These data reveal for the first time that αvβ8 integrin, via interactions with RhoGDI1, regulates activation of Rho proteins to promote GBM cell invasiveness. Hence targeting the αvβ8 integrin–RhoGDI1 signaling axis might be an effective strategy for blocking GBM cell invasion.     

Identification of MYO18A as a Novel Interacting Partner of the PAK2/βPIX/GIT1 Complex and Its Potential Function in Modulating Epithelial Cell Migration

The p21-activated kinase (PAK) 2 is known to be involved in numerous biological functions, including the regulation of actin reorganization and cell motility. To better understand the mechanisms underlying this regulation, we herein used a proteomic approach to identify PAK2-interacting proteins in human epidermoid carcinoma A431 cells. We found that MYO18A, an emerging member of the myosin superfamily, is a novel PAK2 binding partner. Using a siRNA knockdown strategy and in vitro binding assay, we discovered that MYO18A binds to PAK2 through the βPIX/GIT1 complex. Under normal conditions, MYO18A and PAK2 colocalized in lamellipodia and membrane ruffles. Interestingly, knockdown of MYO18A in cells did not prevent formation of the PAK2/βPIX/GIT1 complex, but rather apparently changed its localization to focal adhesions. Moreover, MYO18A-depleted cells showed dramatic changes in morphology and actin stress fiber and membrane ruffle formation and displayed increases in the number and size of focal adhesions. Migration assays revealed that MYO18A-depleted cells had decreased cell motility, and reexpression of MYO18A restored their migration ability. Collectively, our findings indicate that MYO18A is a novel binding partner of the PAK2/βPIX/GIT1 complex and suggest that MYO18A may play an important role in regulating epithelial cell migration via affecting multiple cell machineries.     

A Role of Kindlin-3 in Integrin αMβ2 Outside-In Signaling and the Syk-Vav1-Rac1/Cdc42 Signaling Axis

Integrins mediate cell-cell and cell-extracellular matrix attachments. Integrins are signaling receptors because their cytoplasmic tails are docking sites for cytoskeletal and signaling proteins. Kindlins are a family of band 4.1-ezrin-radixin-moesin-containing intracellular proteins. Apart from regulating integrin ligand-binding affinity, recent evidence suggests that kindlins are involved in integrin outside-in signaling. Kindlin-3 is expressed in platelets, hematopoietic cells and endothelial cells. In humans, loss of kindlin-3 expression accounts for the rare autosomal disease leukocyte adhesion deficiency (LAD) type III that is characterized by bleeding disorders and defective recruitment of leukocytes into sites of infection. Studies have shown that the loss of kindlin-3 expression leads to poor ligand-binding properties of β1, β2 and β3 integrin subfamilies. The leukocyte-restricted β2 integrin subfamily comprises four members, namely αLβ2, αMβ2, αXβ2 and αDβ2. Integrin αMβ2 mediates leukocyte adhesion, phagocytosis, degranulation and it is involved in the maintenance of immune tolerance. Here we provide further evidence that kindlin-3 is required for integrin αMβ2-mediated cell adhesion and spreading using transfected K562 cells that expressed endogenous kindlin-3 but not β2 integrins. K562 stable cell line expressing si-RNA targeting kindlin-3, but not control-si-RNA, and transfected with constitutively activated integrin αMβ2N329S adhered and spread poorly on iC3b. We also show that kindlin-3 is required for the integrin αMβ2-Syk-Vav1 signaling axis that regulates Rac1 and Cdc42 activities. These findings reinforce a role for kindlin-3 in integrin outside-in signaling.     

Binding of the extreme carboxyl-terminus of PAK-interacting exchange factor β (βPIX) to myosin 18A (MYO18A) is required for epithelial cell migration

The PAK2/βPIX/GIT1 (p21-activated kinase 2/PAK-interacting exchange factor-β/G protein-coupled receptor kinase-interactor 1) complex has been shown to distribute to both membrane ruffles and focal adhesions of cells, where it plays an important role in regulating focal adhesion turnover. However, the detailed mechanism underlying this regulation is largely unknown. We previously reported that MYO18Aα interacts via its carboxyl terminus with the PAK2/βPIX/GIT1 complex through direct binding to βPIX, and that knockdown of MYO18Aα in epithelial cells causes accumulation of the complex in focal adhesions and decreased cell migration ability (Hsu et al., 2010). The current study characterized the detailed MYO18Aα–βPIX interaction mechanism and the biological significance of this interaction. We found that deletion of the carboxyl-terminal globular domain of MYO18Aα profoundly altered the cellular localization of βPIX and inhibited cell migration. βPIX interacts through its most carboxyl-terminus, PAWDETNL (639–646), with MYO18Aα and partially colocalized with MYO18Aα in membrane ruffles of cells, whereas βPIX^1–638, a mutant with deletion of PAWDETNL, accumulated in focal adhesions. Both focal adhesion numbers and area in βPIX^1–638-expressing cells were greater than those in cells expressing wild-type βPIX^FL. Further experiments using deletion mutants of MYO18A and βPIX showed that disruption of MYO18A–βPIX interaction not only impaired cell motility but also decreased Rac1 activity. Collectively, our data unravel the interaction regions between MYO18A and βPIX and provide evidence for the critical role of this interaction in regulating cellular localization of βPIX, Rac1 activity, and adhesion and migration in epithelial cells.     

A single nucleotide polymorphism in the translation elongation factor 1α gene correlates with the ability to produce fumonisin in Japanese Fusarium fujikuroi

PCR–RFLP based on the translation elongation factor 1α (TEF) gene was developed to identify Fusarium fujikuroi in the Fusarium (Gibberella) fujikuroi species complex. Ninety-three strains, most of which were obtained from various sources in Japan, were identified as F. fujikuroi and their capability to produce fumonisin was investigated using an in vitro assay. Fumonisin production was detected in 50 strains isolated from maize, strawberry, wheat, and rice, whereas it was undetectable in 43 strains derived from rice seeds and rice seedlings carrying the bakanae disease, and from unknown sources. A single nucleotide polymorphism in the TEF gene (T618G) correlated with the ability to synthesize fumonisin.     

Hmgn1 acts downstream of C/EBPβ to regulate the decidualization of uterine stromal cells in mice

Although Hmgn1 is involved in the regulation of gene expression and cellular differentiation, its physiological roles on the differentiation of uterine stromal cells during decidualization still remain unknown. Here we showed that Hmgn1 mRNA was highly expressed in the decidua on days 6-8 of pregnancy. Simultaneously, increased expression of Hmgn1 was also observed in the artificial and in vitro induced decidualization models. Hmgn1 induced the proliferation of uterine stromal cells and expression of Ccna1, Ccnb1, Ccnb2 and Cdk1 in the absence of estrogen and progesterone. Overexpression of Hmgn1 could enhance the expression of Prl8a2 and Prl3c1 which were 2 well-known differentiation markers for decidualization, whereas inhibition of Hmgn1 with specific siRNA could reduce their expression. Further studies found that Hmgn1 could mediate the effects of C/EBPβ on the expression of Prl8a2 and Prl3c1 during in vitro decidualization. In the uterine stromal cells, cAMP analog 8-Br-cAMP could stimulate the expression of Hmgn1 via C/EBPβ. Moreover, siRNA-mediated down-regulation of Hmgn1 could attenuate the effects of cAMP on the differentiation of uterine stromal cells. During in vitro decidualization, Hmgn1 might act downstream of C/EBPβ to regulate the expression of Cox-2, mPGES-1 and Vegf. Progesterone could up-regulate the expression of Hmgn1 in the ovariectomized mouse uterus, uterine epithelial cells and stromal cells. Knockdown of C/EBPβ with siRNA alleviated the up-regulation of progesterone on Hmgn1 expression. Collectively, Hmgn1 may play an important role during mouse decidualization.     

Entropy as a factor in the binding of γ-aminobutyric acid and nipecotic acid to the γ-aminobutyric acid transport system

Nipecotic acid is one of the most potent competitive inhibitors and alternative substrates for the high-affinity -aminobutyric acid transport system in neurons, but the structural basis of this potency is unclear. Because -aminobutyrate is a highly flexible molecule in solution, it would be expected to lose rotational entropy upon binding to the transport system, a change which does not favor binding. Nipecotic acid, in contrast, is a much less flexible molecule, and one would expect the loss of conformational entropy upon binding to be smaller thus favoring the binding of nipecotic acid over -aminobutyric acid. To investigate this possibility, the thermodynamic parameters, G°, H°, and S°, were determined for the binding of -aminobutyrate and nipecotic acid to the high affinity GABA transport system in synaptosomes. In keeping with expectations, the apparent entropy change for nipecotic acid binding (112±13 J·K^–1) was more favorable than the apparent entropy change for -aminobutyric acid binding (61.3±6.6 J·K^–1). The results suggest that restricted conformation per se is an important contributory factor to the affinity of nipecotic acid for the high-affinity transport system for -aminobutyric acid.This work was conducted when both authors were at the Department of Chemistry, University of Maryland, College Park.Special issue dedicated to Dr. Elling Kvamme.     

Molecular Simulation to Aid in the Understanding of the Aβ(1–42) Peptide of Alzheimer's Disease

Abstract

The Aβ(1–42) peptide of Alzheimer's disease was studied by molecular modeling. The coordinates of the peptide were experimentally generated from solution-NMR spectroscopy, and the conformations were energy minimized using a combination of connectivity-based iterative partial equalization of orbital electronegativity with the MM + force field.

There is a central folded domain in the Aβ peptide. This part is an apolar α-helix. The remaining residues form β-sheets. Aggregation requires that β-sheets interact by noncovalent bonding forces. The unsoluble, aggregated complexes are energetically stable and have ordered structures.

A perspective in drug research is to design compounds that inhibit the hydrophobic cores of the individual Aβ peptides, blocking so the associations between the β-strains.     

β-Carotene as a high-potency antioxidant to prevent the formation of phospholipid hydroperoxides in red blood cells of mice

In order to investigate the antioxidant effect of β-carotene in vivo, phospholipid hydroperoxides and β-carotene isomers in red blood cells (RBC), plasma and tissue organelles were quantitatively measured after the oral administration of β-carotene (94.8% all-trans-β-carotene) to mice. Three groups of 24 mice each were fed for 1 week on a semisynthetic diet supplemented with either 0.6% or 3.0% β-carotene/diet or maintained on a control (β-carotene-unsupplemented) diet. The RBC phospholipid hydroperoxides showed a significant decrease followed by an increase of β-carotene intakes; i.e., 201, 16 and 4 pmol of phosphatidylcholine hydroperoxide/ml packed RBC, and 108, 22 and 8 pmol of phosphatidylethanolamine hydroperoxide/ml packed RBC, in the mice given the control diet, 0.6% carotene diet and 3.0% carotene diet, respectively. The RBC β-carotene increased from 14 to 43 pmol/ml packed RBC as followed by the increase of β-carotene intakes. Such a potent antioxidant effect of β-carotene as observed in RBC was not confirmed in the plasma, liver or lungs, although their β-carotene contents increased. The β-carotene ingestion increased the all-trans-β-carotene d and retinol contents in RBC, plasma, liver and lungs, but the α-tocopherol content decreased. In the β-carotene-supplemented (6 g and 30 g/kg diet) mice, cis-β-carotene content was relatively higher in the RBC (25–35% of total β-carotene) than that in plasma, liver and lungs. The present findings indicate that not only does β-carotene act as a potent antioxidant in vivo but also its antioxidant effect is very specific in the RBC phospholipid bilayers rather than in the plasma and other tissue organelles.