Paralemmin, a prenyl-palmitoyl-anchored phosphoprotein abundant in neurons and implicated in plasma membrane dynamics and cell process formation

We report the identification and initial characterization of paralemmin, a putative new morphoregulatory protein associated with the plasma membrane. Paralemmin is highly expressed in the brain but also less abundantly in many other tissues and cell types. cDNAs from chicken, human, and mouse predict acidic proteins of 42 kD that display a pattern of sequence cassettes with high inter-species conservation separated by poorly conserved linker sequences. Prenylation and palmitoylation of a COOH-terminal cluster of three cysteine residues confers hydrophobicity and membrane association to paralemmin. Paralemmin is also phosphorylated, and its mRNA is differentially spliced in a tissue-specific and developmentally regulated manner. Differential splicing, lipidation, and phosphorylation contribute to electrophoretic heterogeneity that results in an array of multiple bands on Western blots, most notably in brain. Paralemmin is associated with the cytoplasmic face of the plasma membranes of postsynaptic specializations, axonal and dendritic processes and perikarya, and also appears to be associated with an intracellular vesicle pool. It does not line the neuronal plasmalemma continuously but in clusters and patches. Its molecular and morphological properties are reminiscent of GAP-43, CAP-23, and MARCKS, proteins implicated in plasma membrane dynamics. Overexpression in several cell lines shows that paralemmin concentrates at sites of plasma membrane activity such as filopodia and microspikes, and induces cell expansion and process formation. The lipidation motif is essential for this morphogenic activity. We propose a function for paralemmin in the control of cell shape, e.g., through an involvement in membrane flow or in membrane-cytoskeleton interaction.     

GBP, an inhibitor of GSK-3, is implicated in Xenopus development and oncogenesis

Dorsal accumulation of beta-catenin in early Xenopus embryos is required for body axis formation. Recent evidence indicates that beta-catenin is dorsally stabilized by the localized inhibition of the kinase Xgsk-3, utilizing a novel Wnt ligand-independent mechanism. Using a two-hybrid screen, we identified GBP, a maternal Xgsk-3-binding protein that is homologous to a T cell protooncogene in three well-conserved domains. GBP inhibits in vivo phosphorylation by Xgsk-3, and ectopic GBP expression induces an axis by stabilizing beta-catenin within Xenopus embryos. Importantly, antisense oligonucleotide depletion of the maternal GBP mRNA demonstrates that GBP is required for the establishment of the dorsal-ventral axis in Xenopus embryos. Our results define a family of GSK-3-binding proteins with roles in development and cell proliferation.     

Chp, a homologue of the GTPase Cdc42Hs, activates the JNK pathway and is implicated in reorganizing the actin cytoskeleton

The p21-activated protein kinases (PAKs) are activated through direct interaction with the GTPases Rac and Cdc42Hs, which are implicated in the control of the mitogen-activated protein kinase (MAP kinase) c-Jun N-terminal kinase (JNK) and the reorganization of the actin cytoskeleton [1-3]. The exact role of the PAK proteins in these signaling pathways is not entirely clear. To elucidate the biological function of Pak2 and to identify its molecular targets, we used a novel two-hybrid system, the Ras recruitment system (RRS), that aims to detect protein-protein interactions at the inner surface of the plasma membrane (described in the accompanying paper by Broder et al. [4]). The Pak2 regulatory domain (PakR) was fused at the carboxyl terminus of a RasL61 mutant protein and screened against a myristoylated rat pituitary cDNA library. Four clones were identified that interact specifically with PakR and three were subsequently shown to encode a previously unknown homologue of the GTPase Cdc42Hs. This approximately 36 kDa protein, designated Chp, exhibits an overall sequence identity to Cdc42Hs of approximately 52%. Chp contains two additional sequences at the amino and carboxyl termini that are not found in any known GTPase. The amino terminus contains a polyproline sequence, typically found in Src homology 3 (SH3)-binding domains, and the carboxyl terminus appears to be important for Pak2 binding. Results from the microinjection of Chp into cells implicated Chp in the induction of lamellipodia and showed that Chp activates the JNK MAP kinase cascade.     

DDC-4, an apoptosis-associated gene, is a secreted frizzled relative

Pyogenic sacro-iliitis (PS) is a rare disease in childhood. Three cases of PS are reported that were difficult to diagnose. Scintigraphy and magnetic resonance imaging (MRI) were useful for diagnosis. One patient suffered from an episode of relapse. Seventeen other cases of PS were reviewed in the literature to investigate the incidence of abnormal imaging findings and various factors in disease relapse. It was found that the incidence of abnormal findings by scintigraphy was significantly higher than that by computed tomography (P = 0.0057). The duration of intravenous antibiotic administration of the relapse group (14.7 +/- 4.7 days) was significantly shorter than that of the non-relapse group (24.3 +/- 10.7 days; P = 0.0376). The statistical analysis suggested that intravenous antibiotic administration is necessary at least for 20 days to prevent a relapse of PS.     

PAK4, a novel effector for Cdc42Hs, is implicated in the reorganization of the actin cytoskeleton and in the formation of filopodia

The GTPases Rac and Cdc42Hs control diverse cellular functions. In addition to being mediators of intracellular signaling cascades, they have important roles in cell morphogenesis and mitogenesis. We have identified a novel PAK-related kinase, PAK4, as a new effector molecule for Cdc42Hs. PAK4 interacts only with the activated form of Cdc42Hs through its GTPase-binding domain (GBD). Co-expression of PAK4 and the constitutively active Cdc42HsV12 causes the redistribution of PAK4 to the brefeldin A-sensitive compartment of the Golgi membrane and the subsequent induction of filopodia and actin polymerization. Importantly, the reorganization of the actin cytoskeleton is dependent on PAK4 kinase activity and on its interaction with Cdc42Hs. Thus, unlike other members of the PAK family, PAK4 provides a novel link between Cdc42Hs and the actin cytoskeleton. The cellular locations of PAK4 and Cdc42Hs suggest a role for the Golgi in cell morphogenesis.     

Mona, a novel hematopoietic-specific adaptor interacting with the macrophage colony-stimulating factor receptor, is implicated in monocyte/macrophage development

The production, survival and function of monocytes and macrophages are regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms. Binding of M-CSF results in Fms autophosphorylation on specific tyrosines that act as docking sites for intracellular signaling molecules containing SH2 domains. Using a yeast two-hybrid screen, we cloned a novel adaptor protein which we called 'Mona' for monocytic adaptor. Mona contains one SH2 domain and two SH3 domains related to the Grb2 adaptor. Accordingly, Mona interacts with activated Fms on phosphorylated Tyr697, which is also the Grb2-binding site. Furthermore, Mona contains a unique proline-rich region located between the SH2 domain and the C-terminal SH3 domain, and is apparently devoid of any catalytic domain. Mona expression is restricted to two hematopoietic tissues: the spleen and the peripheral blood mononuclear cells, and is induced rapidly during monocytic differentiation of the myeloid NFS-60 cell line in response to M-CSF. Strikingly, overexpression of Mona in bone marrow cells results in strong reduction of M-CSF-dependent macrophage production in vitro. Taken together, our results suggest an important role for Mona in the regulation of monocyte/macrophage development as controlled by M-CSF.     

Slug, a zinc finger gene previously implicated in the early patterning of the mesoderm and the neural crest, is also involved in chick limb development

The great advances made over the last few years in the identification of signalling molecules that pattern the limb bud along the three axes make the limb an excellent model system with which to study developmental mechanisms in vertebrates. The understanding of the signalling networks and their mutual interactions during limb development requires the characterisation of the corresponding downstream genes. In this study we report the expression pattern of Slug, a zinc-finger-containing gene of the snail family, during the development of the limb, and its regulation by distinct axial signalling systems. Slug expression is highly dynamic, and at different stages of limb development can be correlated with the zone of polarizing activity, the progress zone and the interdigital areas. We show that the maintenance of its expression is dependent on signals from the apical ectodermal ridge and independent of Sonic Hedgehog. We also report that, in the interdigit, apoptotic cells lie outside of the domains of Slug expression. The correlation of Slug expression with areas of undifferentiated mesenchyme at stages of tissue differentiation is consistent with its role in early development, in maintaining the mesenchymal phenotype and repressing differentiation processes. We suggest that Slug is involved in the epithelial-mesenchymal interactions that lead to the maintenance of the progress zone.     

KARP-1: a novel leucine zipper protein expressed from the Ku86 autoantigen locus is implicated in the control of DNA-dependent protein kinase activity

The Ku autoantigen plays an integral role in mammalian DNA double-strand break repair as the DNA binding component of the DNA-dependent protein kinase (DNA-PK) complex. Here, we demonstrate that a second gene, KARP-1 (Ku86 Autoantigen Related Protein-1), is expressed from the Ku86 locus. The KARP-1 gene utilizes an upstream promoter and additional exons which results in an extra 9 kDa of protein appended onto the normal Ku86 polypeptide. The KARP-1-specific domain encodes interdigitating hexa- and penta-heptad repeats of leucine residues flanked by a very basic region. Intriguingly, the catalytic subunit of DNA-PK also contains a hexa-heptad repeat of leucines. Consistent with this observation, we observed that human cell lines stably expressing dominant-negative constructs of KARP-1 resulted in diminished DNA-PK activity and X-ray hypersensitivity and that a KARP-1 antibody significantly neutralized DNA-PK activity in vitro. Finally, we present data which suggests that KARP-1 may be primate-specific. These observations have important repercussions for mammalian DNA double-strand break repair.     

Developmental control elements in the promoter of a Drosophila vitelline membrane gene

DNA fragments from the 5' flanking region of Drosophila melanogaster vitelline membrane gene VM26A1 were fused to hsp/lacZ reporter genes, reintroduced via P-element-mediated germ line transformation, and functionally assayed by histological staining for beta-galactosidase activity. A developmental control element capable of promoting the basic vitelline membrane (VM) expression pattern was found within a 176-bp region. Additional multiple genetic control elements were found to be involved in modifying the level and spatial pattern of reporter gene expression but these required the 176-bp element in order to function. Unexpectedly, in isolation this 176-bp core element also promotes abnormal expression of the reporter gene in border cells of stage 8 and 9 egg chambers. This abnormal border cell expression is apparently suppressed in the native gene by an adjacent negative control element.     

mu-Opioid receptor activates signaling pathways implicated in cell survival and translational control

The mu-opioid receptor mediates the analgesic and addictive properties of morphine. Despite the clinical importance of this G-protein-coupled receptor and many years of pharmacological research, few intracellular signaling mechanisms triggered by morphine and other mu-opioid agonists have been described. We report that mu-opioid agonists stimulate three different effectors of a phosphoinositide 3-kinase (PI3K)-dependent signaling cascade. By using a cell line stably transfected with the mu-opioid receptor cDNA, we show that the specific agonist [D-Ala2,N-Me-Phe4,Gly5-ol]enkephalin (DAMGO) stimulates the activity of Akt, a serine/threonine protein kinase implicated in protecting neurons from apoptosis. Activation of Akt by DAMGO correlates with its phosphorylation at serine 473. The selective PI3K inhibitors wortmannin and LY294002 blocked phosphorylation of this site, previously shown to be necessary for Akt enzymatic activity. DAMGO also stimulates the phosphorylation of two other downstream effectors of PI3K, the p70 S6 kinase and the repressors of mRNA translation, 4E-BP1 and 4E-BP2. Upon mu-opioid receptor stimulation, p70 S6 kinase is activated and phosphorylated at threonine 389 and at threonine 421/serine 424. Phosphorylation of p70 S6 kinase and 4E-BP1 is also repressed by PI3K inhibitors as well as by rapamycin, the selective inhibitor of FRAP/mTOR. Consistent with these findings, DAMGO-stimulated phosphorylation of 4E-BP1 impairs its ability to bind the translation initiation factor eIF-4E. These results demonstrate that the mu-opioid receptor activates signaling pathways associated with neuronal survival and translational control, two processes implicated in neuronal development and synaptic plasticity.     

The Drosophila gene alien is expressed in the muscle attachment sites during embryogenesis and encodes a protein highly conserved between plants, Drosophila and vertebrates

1. The mechanisms underlying long-term depression (LTD) of gamma-aminobutyric acid-A (GABAA) receptor-mediated synaptic transmission induced by 10-Hz stimulation of the inhibitory afferents were investigated using perforated and whole cell voltage-clamp recordings from neurons of the deep cerebellar nuclei (DCN). 2. LTD of inhibitory postsynaptic currents (IPSCs) was reliably induced when the 10-Hz stimulation was delivered under current-clamp conditions where the postsynaptic neuronal membrane was allowed to depolarize. 3. Currents elicited by local applications of the GABAA receptor agonist, 4,5,6,7-tetrahydroisoxazolo [5,4-c]pyridin-3-ol hydrochloride (THIP) were also depressed during LTD. 4. LTD could be induced heterosynaptically and did not require the activation of GABAA receptors during the 10-Hz stimulation. 5. In cells loaded with QX-314 and superfused with media containing 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 2-amino-5-phosphonovaleric acid (APV), a series of depolarizing pulses (50 mV, 200 ms) induced a sustained depression of the IPSC. However, this was not observed in cells recorded with high bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA)-containing pipette solutions or when they were exposed to the L-type Ca2+ channel antagonist, nitrendipine. 6. The 10-Hz-induced LTD was also inhibited by BAPTA and was significantly reduced when DCN cells were loaded with microcystin LR or treated with okadaic acid, both inhibitors of protein phosphatases. 7. These results indicate that increases in postsynaptic [Ca2+] and phosphatase activity can reduce the efficacy of GABAA receptor-mediated synaptic transmission.     

KIF2beta, a new kinesin superfamily protein in non-neuronal cells, is associated with lysosomes and may be implicated in their centrifugal translocation

Lysosomes concentrate juxtanuclearly in the region around the microtubule-organizing center by interaction with microtubules. Different experimental and physiological conditions can induce these organelles to move to the cell periphery by a mechanism implying a plus-end-directed microtubule-motor protein (a kinesin-like motor). The responsible kinesin-superfamily protein, however, is unknown. We have identified a new mouse isoform of the kinesin superfamily, KIF2beta, an alternatively spliced isoform of the known, neuronal kinesin, KIF2. Developmental expression pattern and cell-type analysis in vivo and in vitro reveal that KIF2beta is abundant at early developmental stages of the hippocampus but is then downregulated in differentiated neuronal cells, and it is mainly or uniquely expressed in non-neuronal cells while KIF2 remains exclusively neuronal. Electron microscopy of mouse fibroblasts and immunofluorescence of KIF2beta-transiently-transfected fibroblasts show KIF2 and KIF2beta primarily associated with lysosomes, and this association can be disrupted by detergent treatment. In KIF2beta-overexpressing cells, lysosomes (labeled with anti-lysosome-associated membrane protein-1) become abnormally large and peripherally located at some distance from their usual perinuclear positions. Overexpression of KIF2 or KIF2beta does not change the size or distribution of early, late and recycling endosomes nor does overexpression of different kinesin superfamily proteins result in changes in lysosome size or positioning. These results implicate KIF2beta as a motor responsible for the peripheral translocation of lysosomes.