GPR55 regulates cannabinoid 2 receptor-mediated responses in human neutrophils

The directional migration of neutrophils towards inflammatory mediators, such as chemokines and cannabinoids, occurs via the activation of seven transmembrane G protein coupled receptors (7TM/GPCRs) and is a highly organized process. A crucial role for controlling neutrophil migration has been ascribed to the cannabinoid CB(2) receptor (CB(2)R), but additional modulatory sites distinct from CB(2)R have recently been suggested to impact CB(2)R-mediated effector functions in neutrophils. Here, we provide evidence that the recently de-orphanized 7TM/GPCR GPR55 potently modulates CB(2)R-mediated responses. We show that GPR55 is expressed in human blood neutrophils and its activation augments the migratory response towards the CB(2)R agonist 2-arachidonoylglycerol (2-AG), while inhibiting neutrophil degranulation and reactive oxygen species (ROS) production. Using HEK293 and HL60 cell lines, along with primary neutrophils, we show that GPR55 and CB(2)R interfere with each other's signaling pathways at the level of small GTPases, such as Rac2 and Cdc42. This ultimately leads to cellular polarization and efficient migration as well as abrogation of degranulation and ROS formation in neutrophils. Therefore, GPR55 limits the tissue-injuring inflammatory responses mediated by CB(2)R, while it synergizes with CB(2)R in recruiting neutrophils to sites of inflammation.     

New pharmacokinetic and pharmacodynamic tools for interferon-alpha (IFN-α) treatment of human cancer

Interferon (IFN-) has been widely used in the treatment of human solid and haematologic malignancies. Although the antitumour activity of IFN- is well recognised at present, no major advances have been achieved in the last few years. Recent findings have provided new information on the molecular mechanisms of the antitumour activity of the cytokine. In fact, IFN- appears to block cell proliferation, at least in part, through the induction of apoptotic effects. This cytokine can also regulate the progression of tumour cells through the different phases of the cell cycle inducing an increase of the expression of the cyclin-dependent kinase inhibitors p21 and p27. However, it must be considered that IFN- is a physiologic molecule with ubiquitously expressed receptors that is likely to activate survival mechanisms in the cell. We have recently identified an epidermal growth factor (EGF) Ras-dependent protective response to the apoptosis induced by IFN- in epidermoid cancer cells. The identification of tissue- and/or tumour-specific survival pathways and their selective targeting might provide a new approach to improve the efficacy of IFN-–based treatment of human cancer. Moreover, new pegylated species of IFN- are now available with a more favourable pharmacokinetic profile. We will review these achievements, and we will specifically address the topic of IFN-–based molecularly targeted combinatory antitumour approaches.     

The integrin α6β1 modulation of PI3K and Cdc42 activities induces dynamic filopodium formation in human platelets

Summary Platelets are an ideal model for studying a rapid morphological change in response to various signal transduction systems. Morphological changes via the activation of integrin αIIbβ3 in platelets have been investigated intensively. In contrast, activation via integrin α6β1 is less well studied. Here, we provide the first biochemical evidence that integrins α6β1 and αIIbβ3 of platelets are associated with different membrane proteins. We also demonstrate that platelets activated by integrin α6β1 show dynamic change by actively forming filopodia and never fully spreading over a period of more than an hour. In addition, platelets activated by integrin α6β1 are different from those activated by integrin αIIbβ3 in terms of cell–substrate contact and in their distribution pattern of actin, Arp2/3 and various phosphotyrosine proteins. The morphological appearance of platelets produced through integrin α6β1 activation is highly dependent on PI3 kinase (PI3K) but less dependent on Src kinase. Suppression of PI3K activity in integrin α6β1 activated platelets induces an increase in Cdc42 activity and more filopodium formation. However, both Cdc42 and PI3K activity are higher in platelets activated by integrin α6β1 than in those activated by integrin αIIbβ3. Taken together, this study demonstrates that the signals induced by integrin α6β1 modulate at the level of PI3K and Cdc42 activity to allow platelets to actively form filopodia.     

Identification and functional characterization of <Emphasis Type="Italic">Candida albicans CDC4</Emphasis>

Summary The CDC4 gene of Saccharomyces cerevisiae encodes an essential function that is required for G1-S and G2-M transitions during mitosis and at various stages during meiosis. We have isolated a functional homologue of CDC4 (CaCDC4) from the pathogenic yeast Candida albicans by complementing the S. cerevisiae cdc4-3 mutation with CaCDC4 expressed from its own promoter on a single-copy vector. The predicted product of CaCDC4 has 37% overall identity to the S. cerevisiae Cdc4 protein, although this identity is biased towards the C-terminal region of the two proteins which contains eight copies of the degenerate WD-40 motif, an element found in proteins that regulate diverse biological processes and an F-box domain proximal to the first iteration of the WD-40 motif. Both the F-box domain and WD-40 motifs appear necessary for the mitotic functions of Cdc4 in both yeasts. In contrast to its conserved role in mitosis, C. albicans CDC4 is unable to rescue the meiotic deficiency in a S. cerevisiae cdc4 homozygous diploid under restrictive conditions, even when expressed from an efficient S. cerevisiae promoter. In opposition to S. cerevisiae CDC4 being essential, C. albicans CDC4 appears to be nonessential and in its absence is critical for filamentous growth in C. albicans.     

Electrostatic contributions to the kinetics and thermodynamics of protein assembly

The role of electrostatic interactions in the assembly of a native protein structure was studied using fragment complementation. Contributions of salt, pH, or surface charges to the kinetics and equilibrium of calbindin D(9k) reconstitution was measured in the presence of Ca(2+) using surface plasmon resonance and isothermal titration calorimetry. Whereas surface charge substitutions primarily affect the dissociation rate constant, the association rates are correlated with subdomain net charge in a way expected for Coulomb interactions. The affinity is reduced in all mutants, with the largest effect (260-fold) observed for the double mutant K25E+K29E. At low net charge, detailed charge distribution is important, and charges remote from the partner EF-hand have less influence than close ones. The effects of salt and pH on the reconstitution are smaller than mutational effects. The interaction between the wild-type EF-hands occurs with high affinity (K(A) = 1.3 x 10(10) M(-1); K(D) = 80 pM). The enthalpy of association is overall favorable and there appears to be a very large favorable entropic contribution from the desolvation of hydrophobic surfaces that become buried in the complex. Electrostatic interactions contribute significantly to the affinity between the subdomains, but other factors, such as hydrophobic interactions, dominate.     

RhoGDIs revisited: novel roles in Rho regulation

Small GTP-binding proteins of the Rho/Rac/Cdc42 family combine their GDP/GTP cycle, regulated by guanine nucleotide-exchange factors and GTPase-activating proteins, to a cytosol/membrane cycle, regulated by guanine nucleotide dissociation inhibitors (rhoGDIs). RhoGDIs are endowed with dual functions in the cytosol where they form soluble complexes with geranylgeranylated GDP-bound Rho proteins and at membrane interfaces where they monitor the delivery and extraction of Rho proteins to/from their site of action. They have little diversity compared with other Rho protein regulators and therefore have been regarded mostly as housekeeping regulators that distribute Rho proteins equally to any membranes. Recently, acquired data show that rhoGDIs, by interacting with candidate receptors/displacement factors or by phosphorylation, may in fact have active contributions to targeting Rho proteins to specific subcellular membranes and signaling pathways. In addition, the GDP/GTP and membrane/cytosol cycles can be uncoupled in certain cases, with Rho proteins either escaping the membrane/cytosol cycle or being regulated by rhoGDIs in their GTP-bound form. Here, we survey recent structure-function relationships and cellular studies on rhoGDIs and revisit their classical housekeeping role into novel and more specific functions. We also review their involvement in diseases.     

Changes of enzyme activity in lipid signaling pathways related to substrate reordering

The static fluid mosaic model of biological membranes has been progressively complemented by a dynamic membrane model that includes phospholipid reordering in domains that are proposed to extend from nanometers to microns. Kinetic models for lipolytic enzymes have only been developed for homogeneous lipid phases. In this work, we develop a generalization of the well-known surface dilution kinetic theory to cases where, in a same lipid phase, both domain and nondomain phases coexist. Our model also allows understanding the changes in enzymatic activity due to a decrease of free substrate concentration when domains are induced by peptides. This lipid reordering and domain dynamics can affect the activity of lipolytic enzymes, and can provide a simple explanation for how basic peptides, with a strong direct interaction with acidic phospholipids (such as beta-amyloid peptide), may cause a complex modulation of the activities of many important enzymes in lipid signaling pathways.     

SNX9 as an adaptor for linking synaptojanin-1 to the Cdc42 effector ACK1

Sorting nexin 9 (SNX9, also referred to as SH3PX1) is a binding partner for the non-receptor and Cdc42-associated kinase (ACK) in Drosophila and mammals. ACK1 is known to bind clathrin and influence EGF receptor endocytosis. SNX9 comprises an N-terminal Src homology domain 3 (SH3), a central PHOX homology (PX) domain, and a carboxyl-terminal coiled-coil region. In order to investigate SNX9 further we have made use of a novel in vivo biotinylation system to label various GST-SH3 domains and perform blot overlays, thereby identifying synaptojanin-1 as a partner for SNX9. Biotinylated SH3 domains were also used for specific identification of target proline-rich sequences in synaptojanin and ACK1 on synthetic peptides arrays. Direct assessment of SH3 binding efficiencies at different positions within the extensive proline-rich regions of these proteins were thus determined. While SNX9 targets a number of sequences within the proline-rich regions of synaptojanin, a single site was identified in human ACK1. By testing the association of various truncations of ACK1 with SNX9 we confirmed the dominant SNX9 binding domain in human ACK1 (residues 920-955). In the presence of SNX9 we find that synaptojanin is able to colocalize with distinct ACK1 containing vesicles, indicating that this tyrosine kinase is linked to many components involved in vesicle dynamics including clathrin, AP2 and synaptojanin-1.     

Mechanisms for HIV Tat upregulation of IL-10 and other cytokine expression: kinase signaling and PKR-mediated immune response

HIV Tat has been known to have multiple regulatory roles including replication of HIV and modulation of cellular kinases. We investigated whether signaling kinase PKR plays a critical role in mediating Tat-induced cytokine dysregulation. We showed Tat induction of IL-10 dysregulation is associated with PKR activation. To examine the mechanism involved, inhibition of PKR activity abrogated the Tat-induced cytokine induction. We next identified that the MAP kinases including ERK-1/2 and p38 are downstream of PKR in these Tat-induced pathways. Thus, PKR may play a critical role in mediating the subversive effects of HIV Tat resulting in IL-10 induction.     

The c-Fes tyrosine kinase cooperates with the breakpoint cluster region protein (Bcr) to induce neurite extension in a Rac- and Cdc42-dependent manner

The c-fes locus encodes a cytoplasmic protein-tyrosine kinase (Fes) previously shown to accelerate nerve growth factor (NGF)-induced neurite outgrowth in rat PC12 cells. Here, we investigated the role of the Rho family small GTPases Rac1 and Cdc42 in Fes-mediated neuritogenesis, which have been implicated in neuronal differentiation in other systems. Fes-induced acceleration of neurite outgrowth in response to NGF treatment was completely blocked by the expression of dominant-negative Rac1 or Cdc42. Expression of a kinase-active mutant of Fes induced constitutive relocalization of endogenous Rac1 to the cell periphery in the absence of NGF, and led to dramatic actin reorganization and spontaneous neurite extension. We also investigated the breakpoint cluster region protein (Bcr), which possesses the Dbl and PH domains characteristic of guanine nucleotide exchange factors for Rho family GTPases, as a possible link between Fes, Rac/Cdc42 activation, and neuritogenesis. Coexpression of a GFP-Bcr fusion protein containing the Fes binding and tyrosine phosphorylation sites (amino acids 162-413) completely suppressed neurite outgrowth triggered by Fes. Conversely, coexpression of full-length Bcr with wild-type Fes in PC12 cells induced NGF-independent neurite formation. Taken together, these data suggest that Fes and Bcr cooperate to activate Rho family GTPases as part of a novel pathway regulating neurite extension in PC12 cells, and provide more evidence for an emerging role for Fes in neuronal differentiation.