The C. elegans Frizzled CFZ-2 is required for cell migration and interacts with multiple Wnt signaling pathways

Members of the Frizzled family of integral membrane proteins are implicated in many developmental events, including specifying cell fate, orienting cell and planar polarity, and directing cell migration. Frizzleds function as cell surface receptors for secreted Wnt proteins. We report here the isolation of a mutation in cfz-2, a Caenorhabditis elegans Frizzled gene. Mutation of cfz-2 causes defective cell migration, disorganization of head neurons, and can cause ectopic axon outgrowth. Analysis of mosaic animals shows that CFZ-2 functions cell nonautonomously, but does not rule out an autonomous role. CFZ-2 is expressed primarily in the anterior of embryos and in several cells in the head of adults. Our analysis of interactions between CFZ-2 and other Wnt pathways reveals that three Wnts, CWN-1, CWN-2 and EGL-20, and a Frizzled, MOM-5, function redundantly with one another and with CFZ-2 for specific cell migrations. In contrast, CWN-1, CWN-2, EGL-20, CFZ-2, and MOM-5 antagonize one another for other migrations. Therefore, CFZ-2 functions by collaborating with and/or antagonizing other Wnt signaling pathways to regulate specific cell migrations.     

Protein kinase C(alpha) is required for vanilloid receptor 1 activation. Evidence for multiple signaling pathways

Activation of vanilloid receptor (VR1) by protein kinase C (PKC) was investigated in cells ectopically expressing VR1 and primary cultures of dorsal root ganglion neurons. Submicromolar phorbol 12,13-dibutyrate (PDBu), which stimulates PKC, acutely activated Ca(2+) uptake in VR1-expressing cells at pH 5.5, but not at mildly acidic or neutral pH. PDBu was antagonized by bisindolylmaleimide, a PKC inhibitor, and ruthenium red, a VR1 ionophore blocker, but not capsazepine, a vanilloid antagonist indicating that catalytic activity of PKC is required for PDBu activation of VR1 ion conductance, and is independent of the vanilloid site. Chronic PDBu dramatically down-regulated PKC(alpha) in dorsal root ganglion neurons or the VR1 cell lines, whereas only partially influencing PKCbeta, -delta, -epsilon, and -zeta. Loss of PKC(alpha) correlated with loss of response to acute re-challenge with PDBu. Anandamide, a VR1 agonist in acidic conditions, acts additively with PDBu and remains effective after chronic PKC down-regulation. Thus, two independent VR1 activation pathways can be discriminated: (i) direct ligand binding (anandamide, vanilloids) or (ii) extracellular ligands coupled to PKC by intracellular signaling. Experiments in cell lines co-expressing VR1 with different sets of PKC isozymes showed that acute PDBu-induced activation requires PKC(alpha), but not PKC(epsilon). These studies suggest that PKC(alpha) in sensory neurons may elicit or enhance pain during inflammation or ischemia.     

Double-stranded RNA-activated protein kinase interacts with apoptosis signal-regulating kinase 1. Implications for apoptosis signaling pathways.

Double-stranded RNA-activated protein kinase (PKR), a serine/threonine kinase, is activated in virus-infected cells and acts as an antiviral machinery of type I interferons. PKR controls several stress response pathways induced by double-stranded RNA, tumor necrosis factor-alpha or lipopolysaccharide, which result in the activation of stress-activated protein kinase/c-Jun NH2-terminal kinase and p38 of the mitogen-activated protein kinase family. Here we showed a novel interaction between PKR and apoptosis signal-regulating kinase 1 (ASK1), one of the members of the mitogen-activated protein kinase kinase kinase family, which is activated in response to a variety of apoptosis-inducing stimuli. PKR and ASK1 showed predominant cytoplasmic localization in COS-1 cells transfected with both cDNAs, and coimmunoprecipitated from the cell extracts. A dominant negative mutant of PKR (PKR-KR) inhibited both the apoptosis and p38 activation induced by ASK1 in vivo. Consistently, PKR-KR inhibited the autophosphorylation of ASK1 in vitro, and exposure to poly(I)-poly(C) increased the phosphorylation of ASK1 in vivo. These results indicate the existence of a link between PKR and ASK1, which modifies downstream MAPK.     

Epac, in synergy with cAMP-dependent protein kinase (PKA), is required for cAMP-mediated mitogenesis

cAMP stimulates proliferation in many cell types. For many years, cAMP-dependent protein kinase (PKA) represented the only known cAMP effector. PKA, however, does not fully mimic the action of cAMP, indicating the existence of a PKA-independent component. Since cAMP-mediated activation of the G-protein Rap1 and its phosphorylation by PKA are strictly required for the effects of cAMP on mitogenesis, we hypothesized that the Rap1 activator Epac might represent the PKA-independent factor. Here we report that Epac acts synergistically with PKA in cAMP-mediated mitogenesis. We have generated a new dominant negative Epac mutant that revealed that activation of Epac is required for thyroid-stimulating hormone or cAMP stimulation of DNA synthesis. We demonstrate that Epac's action on cAMP-mediated activation of Rap1 and cAMP-mediated mitogenesis depends on the subcellular localization of Epac via its DEP domain. Disruption of the DEP-dependent subcellular targeting of Epac abolished cAMP-Epac-mediated Rap1 activation and thyroid-stimulating hormone-mediated cell proliferation, indicating that an Epac-Rap-PKA signaling unit is critical for the mitogenic action of cAMP.     

The human microcephaly protein STIL interacts with CPAP and is required for procentriole formation

Centriole duplication involves the growth of a procentriole next to the parental centriole. Mutations in STIL and CPAP/CENPJ cause primary microcephaly (MCPH). Here, we show that human STIL has an asymmetric localization to the daughter centriole and is required for procentriole formation. STIL levels oscillate during the cell cycle. Interestingly, STIL interacts directly with CPAP and forms a complex with hSAS6. A natural mutation of CPAP (E1235V) that causes MCPH in humans leads to significantly lower binding to STIL. Overexpression of STIL induced the formation of multiple procentrioles around the parental centriole. STIL depletion inhibited normal centriole duplication, Plk4-induced centriole amplification, and CPAP-induced centriole elongation, and resulted in a failure to localize hSAS6 and CPAP to the base of the nascent procentriole. Furthermore, hSAS6 depletion hindered STIL targeting to the procentriole, implying that STIL and hSAS6 are mutually dependent for their centriolar localization. Together, our results indicate that the two MCPH-associated proteins STIL and CPAP interact with each other and are required for procentriole formation, implying a central role of centriole biogenesis in MCPH.     

A rho-binding protein kinase C-like activity is required for the function of protein kinase N in Drosophila development

The Rho GTPases interact with multiple downstream effectors to exert their biological functions, which include important roles in tissue morphogenesis during the development of multicellular organisms. Among the Rho effectors are the protein kinase N (PKN) proteins, which are protein kinase C (PKC)-like kinases that bind activated Rho GTPases. The PKN proteins are well conserved evolutionarily, but their biological role in any organism is poorly understood. We previously determined that the single Drosophila ortholog of mammalian PKN proteins, Pkn, is a Rho/Rac-binding kinase essential for Drosophila development. By performing "rescue" studies with various Pkn mutant constructs, we have defined the domains of Pkn required for its role during Drosophila development. These studies suggested that Rho, but not Rac binding is important for Pkn function in development. In addition, we determined that the kinase domain of PKC53E, a PKC family kinase, can functionally substitute for the kinase domain of Pkn during development, thereby exemplifying the evolutionary strategy of "combining" functional domains to produce proteins with distinct biological activities. Interestingly, we also identified a requirement for Pkn in wing morphogenesis, thereby revealing the first postembryonic function for Pkn.     

A Dictyostelium SH2 adaptor protein required for correct DIF-1 signaling and pattern formation

Dictyostelium is the only non-metazoan with functionally analyzed SH2 domains and studying them can give insights into their evolution and wider potential. LrrB has a novel domain configuration with leucine-rich repeat, 14-3-3 and SH2 protein–protein interaction modules. It is required for the correct expression of several specific genes in early development and here we characterize its role in later, multicellular development. During development in the light, slug formation in LrrB null (lrrB-) mutants is delayed relative to the parental strain, and the slugs are highly defective in phototaxis and thermotaxis. In the dark the mutant arrests development as an elongated mound, in a hitherto unreported process we term dark stalling. The developmental and phototaxis defects are cell autonomous and marker analysis shows that the pstO prestalk sub-region of the slug is aberrant in the lrrB- mutant. Expression profiling, by parallel micro-array and deep RNA sequence analyses, reveals many other alterations in prestalk-specific gene expression in lrrB- slugs, including reduced expression of the ecmB gene and elevated expression of ampA. During culmination ampA is ectopically expressed in the stalk, there is no expression of ampA and ecmB in the lower cup and the mutant fruiting bodies lack a basal disc. The basal disc cup derives from the pstB cells and this population is greatly reduced in the lrrB- mutant. This anatomical feature is a hallmark of mutants aberrant in signaling by DIF-1, the polyketide that induces prestalk and stalk cell differentiation. In a DIF-1 induction assay the lrrB- mutant is profoundly defective in ecmB activation but only marginally defective in ecmA induction. Thus the mutation partially uncouples these two inductive events. In early development LrrB interacts physically and functionally with CldA, another SH2 domain containing protein. However, the CldA null mutant does not phenocopy the lrrB- in its aberrant multicellular development or phototaxis defect, implying that the early and late functions of LrrB are affected in different ways. These observations, coupled with its domain structure, suggest that LrrB is an SH2 adaptor protein active in diverse developmental signaling pathways.     

Drosophila sticky/citron kinase is a regulator of cell-cycle progression, genetically interacts with Argonaute 1 and modulates epigenetic gene silencing

The sticky/citron kinase protein is a conserved regulator of cell-cycle progression from invertebrates to humans. While this kinase is essential for completion of cytokinesis, sticky/citron kinase phenotypes disrupting neurogenesis and cell differentiation suggest additional non-cell-cycle functions. However, it is not known whether these phenotypes are an indirect consequence of sticky mutant cell-cycle defects or whether they define a novel function for this kinase. We have isolated a temperature-sensitive allele of the Drosophila sticky gene and we show that sticky/citron kinase is required for histone H3-K9 methylation, HP1 localization, and heterochromatin-mediated gene silencing. sticky genetically interacts with Argonaute 1 and sticky mutants exhibit context-dependent Su(var) and E(var) activity. These observations indicate that sticky/citron kinase functions to regulate both actin-myosin-mediated cytokinesis and epigenetic gene silencing, possibly linking cell-cycle progression to heterochromatin assembly and inheritance of gene expression states.     

Drosophila chibby is required for basal body formation and ciliogenesis but not for Wg signaling

Centriole-to-basal body conversion, a complex process essential for ciliogenesis, involves the progressive addition of specific proteins to centrioles. CHIBBY (CBY) is a coiled-coil domain protein first described as interacting with β-catenin and involved in Wg-Int (WNT) signaling. We found that, in Drosophila melanogaster, CBY was exclusively expressed in cells that require functional basal bodies, i.e., sensory neurons and male germ cells. CBY was associated with the basal body transition zone (TZ) in these two cell types. Inactivation of cby led to defects in sensory transduction and in spermatogenesis. Loss of CBY resulted in altered ciliary trafficking into neuronal cilia, irregular deposition of proteins on spermatocyte basal bodies, and, consequently, distorted axonemal assembly. Importantly, cby(1/1) flies did not show Wingless signaling defects. Hence, CBY is essential for normal basal body structure and function in Drosophila, potentially through effects on the TZ. The function of CBY in WNT signaling in vertebrates has either been acquired during vertebrate evolution or lost in Drosophila.     

The Bax N terminus is required for negative regulation by the mitogen-activated protein kinase kinase and Akt signaling pathways in T cells

The Bcl-2 family proapoptotic protein, Bax, redistributes to the mitochondrion in response to varied stimuli, triggering loss of mitochondrial integrity and apoptosis. Suppression of MAPK kinase (MEK1) by the reagent UO126 in activated T cells maintained in the cytokine IL-2 disrupts cytoplasmic localization of Bax and cell survival. UO126 triggers mitochondrial translocation of ectopically expressed Bax-GFP, and both UO126 and dominant negative MEK-1 (DN-MEK1) trigger increased apoptosis in Bax-GFP-expressing T cell lines. Because inhibition of PI3K or its target Akt also triggers mitochondrial translocation of Bax in T cells and apoptosis in Bax-transfected cell lines, we generated Bax deletion mutants to identify the region(s) that confers sensitivity to regulation by MEK1 and Akt. A deletion mutant (Bax(1-171)) without the C terminus mitochondrial targeting sequence or an Akt target site (Ser(184)) localizes to the cytoplasm and triggers low level apoptosis that is enhanced by DN-Akt or DN-MEK1. A construct that lacks the first 29 aa (Bax-delta29) largely localizes to mitochondria, is highly apoptogenic, and is not inhibited by Akt or MEK1. Furthermore, Bax-delta29 overcomes IL-2-dependent survival in a T cell line, whereas Bax triggers comparatively low levels of apoptosis in these cells. Cytoplasmic localization and regulation by MEK1 and Akt are restored in a mutant deleted of the first 13 aa (Bax-delta13). Taken together, our results identify a region in the Bax N terminus that determines cellular localization regulated by MEK- and Akt-dependent signaling in T cells.     

The Drosophila Lkb1 kinase is required for spindle formation and asymmetric neuroblast division

We have isolated lethal mutations in the Drosophila lkb1 gene (dlkb1), the homolog of C. elegans par-4 and human LKB1 (STK11), which is mutated in Peutz-Jeghers syndrome. We show that these mutations disrupt spindle formation, resulting in frequent polyploid cells in larval brains. In addition, dlkb1 mutations affect asymmetric division of larval neuroblasts (NBs); they suppress unequal cytokinesis, abrogate proper localization of Bazooka, Par-6, DaPKC and Miranda, but affect neither Pins/Galphai localization nor spindle rotation. Most aspects of the dlkb1 phenotype are exacerbated in dlkb1 pins double mutants, which exhibit more severe defects than those observed in either single mutant. This suggests that Dlkb1 and Pins act in partially redundant pathways to control the asymmetry of NB divisions. Our results also indicate that Dlkb1 and Pins function in parallel pathways controlling the stability of spindle microtubules. The finding that Dlkb1 mediates both the geometry of stem cell division and chromosome segregation provides novel insight into the mechanisms underlying tumor formation in Peutz-Jeghers patients.     

A RanGAP protein physically interacts with the NB-LRR protein Rx, and is required for Rx-mediated viral resistance

Race-specific disease resistance in plants is mediated by the products of host disease resistance (R) genes. Plant genomes possess hundreds of R gene homologs encoding nucleotide-binding and leucine-rich repeat (NB-LRR) proteins. NB-LRR proteins induce a disease resistance response following recognition of pathogen-encoded avirulence (Avr) proteins. However, little is known about the general mechanisms by which NB-LRR proteins recognize Avr proteins or how they subsequently induce defense responses. The Rx NB-LRR protein of potato confers resistance to potato virus X (PVX). Using a co-purification strategy, we have identified a Ran GTPase-activating protein (RanGAP2) as an Rx-interacting protein. We show by co-immunoprecipitation that this interaction is mediated in planta through the putative signaling domain at the Rx amino terminus. Overexpression of RanGAP2 results in activation of certain Rx derivatives. Likewise, knocking down RanGAP2 expression in Nicotiana benthamiana by virus-induced gene silencing compromises Rx-mediated resistance to PVX. Thus, we have demonstrated a novel role for a RanGAP in the function of a plant disease resistance response.     

The Aspergillus nidulans putative kinase, KfsA (kinase for septation), plays a role in septation and is required for efficient asexual spore formation

In Aspergillus nidulans nuclear division and cytokinesis are coupled processes during asexual sporulation. Metulae, phialides and conidia contain a single nucleus. Here we describe the role of a putative Saccharomyces cerevisiae Kin4-related kinase, KfsA (kinase for septation) in the control of septum formation in A. nidulans. The kfsA deletion caused an increase in the number of conidiophores with septa in their stalks from 20% in wild type to 60% in the mutant strain. Interestingly, 7% of metulae contained two nuclei and the corresponding phialides remained anucleate, suggesting septum formation before proper segregation of nuclei. This points to a checkpoint control of KfsA, which prevents septum formation before nuclear separation. KfsA localized to the cortex and septa in hyphae and in conidiophores but not to the spindle-pole bodies, as it was shown for Kin4 in yeast. KfsA appeared at septa after actin disappeared, suggesting an additional role of KfsA late during septum formation.     

A Drosophila orthologue of larp protein family is required for multiple processes in male meiosis

It is important for the proper execution of cell division in both mitosis and meiosis that the chromosome segregation, cytokinesis, and partition of cell organelles progress in smooth coordination. We show here that the mitochondria inheritance is closely linked with microtubules during meiotic divisions in Drosophila males. They are first clustered in a cell equator at metaphase associated with astral microtubules and then distributed along central spindle microtubules after anaphase. The molecular mechanism for the microtubule-dependent inheritance of mitochondria in male meiosis has not been demonstrated yet. We first isolated mutations for a larp gene that is highly conserved among eukaryotes and showed that these mutant males exhibited multiple meiotic phenotypes such as a failure of chromosome segregation, cytokinesis, and mitochondrial partition. Our cytological examination revealed that the mutants showed defects in spindle pole organization and spindle formation. The larp encodes a Drosophila orthologue of a La-related protein containing a domain exhibiting an outstanding homology with a La type RNA-binding protein. Surprisingly, the dLarp protein is localized in the cytoplasm of the male germ line cells, as observed by its distinct co-localization with mitochondria in early spermatocytes and during meiotic divisions. We discuss here the essential role that dLarp plays in multiple processes in Drosophila male meiosis.     

A protein kinase from Colletotrichum trifolii is induced by plant cutin and is required for appressorium formation

When certain phytopathogenic fungi contact plant surfaces, specialized infection structures (appressoria) are produced that facilitate penetration of the plant external barrier; the cuticle. Recognition of this hydrophobic host surface must be sensed by the fungus, initiating the appropriate signaling pathway or pathways for pathogenic development. Using polymerase chain reaction and primers designed from mammalian protein kinase C sequences (PKC), we have isolated, cloned, and characterized a protein kinase from Colletotrichum trifolii, causal agent of alfalfa anthracnose. Though sequence analysis indicated conserved sequences in mammalian PKC genes, we were unable to induce activity of the fungal protein using known activators of PKC. Instead, we show that the C. trifolii gene, designated LIPK (lipid-induced protein kinase) is induced specifically by purified plant cutin or long-chain fatty acids which are monomeric constituents of cutin. PKC inhibitors prevented appressorium formation and, to a lesser extent, spore germination. Overexpression of LIPK resulted in multiple, abnormally shaped appressoria. Gene replacement of lipk yielded strains which were unable to develop appressoria and were unable to infect intact host plant tissue. However, these mutants were able to colonize host tissue following artificial wounding, resulting in typical anthracnose lesions. Taken together, these data indicate a central role in triggering infection structure formation for this protein kinase, which is induced specifically by components of the plant cuticle. Thus, the fungus is able to sense and use host surface chemistry to induce a protein kinase-mediated pathway that is required for pathogenic development.