Phosphorylation of Serine Palmitoyltransferase Long Chain-1 (SPTLC1) on Tyrosine 164 Inhibits Its Activity and Promotes Cell Survival

In BCR-ABL-expressing cells, sphingolipid metabolism is altered. Because the first step of sphingolipid biosynthesis occurs in the endoplasmic reticulum (ER), our objective was to identify ABL targets in the ER. A phosphoproteomic analysis of canine pancreatic ER microsomes identified 49 high scoring phosphotyrosine-containing peptides. These were then categorized in silico and validated in vitro. We demonstrated that the ER-resident human protein serine palmitoyltransferase long chain-1 (SPTLC1), which is the first enzyme of sphingolipid biosynthesis, is phosphorylated at Tyr¹⁶⁴ by the tyrosine kinase ABL. Inhibition of BCR-ABL using either imatinib or shRNA-mediated silencing led to the activation of SPTLC1 and to increased apoptosis in both K562 and LAMA-84 cells. Finally, we demonstrated that mutation of Tyr¹⁶⁴ to Phe in SPTLC1 increased serine palmitoyltransferase activity. The Y164F mutation also promoted the remodeling of cellular sphingolipid content, thereby sensitizing K562 cells to apoptosis. Our observations provide a mechanistic explanation for imatinib-mediated cell death and a novel avenue for therapeutic strategies.     

The SPTLC3 Subunit of Serine Palmitoyltransferase Generates Short Chain Sphingoid Bases

The enzyme serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in the de novo synthesis of sphingolipids. Previously the mammalian SPT was described as a heterodimer composed of two subunits, SPTLC1 and SPTLC2. Recently we identified a novel third SPT subunit (SPTLC3). SPTLC3 shows about 68% identity to SPTLC2 and also includes a pyridoxal phosphate consensus motif. Here we report that the overexpression of SPTLC3 in HEK293 cells leads to the formation of two new sphingoid base metabolites, namely C₁₆-sphinganine and C₁₆-sphingosine. SPTLC3-expressing cells have higher in vitro SPT activities with lauryl- and myristoyl-CoA than SPTLC2-expressing cells, and SPTLC3 mRNA expression levels correlate closely with the C₁₆-sphinganine synthesis rates in various human and murine cell lines. Approximately 15% of the total sphingolipids in human plasma contain a C₁₆ backbone and are found in the high density and low density but not the very low density lipoprotein fraction. In conclusion, we show that the SPTLC3 subunit generates C₁₆-sphingoid bases and that sphingolipids with a C₁₆ backbone constitute a significant proportion of human plasma sphingolipids.Sphingolipids comprise a class of bioactive lipids that contribute to plasma membrane and plasma lipoprotein formation and exert a broad range of cellular signaling functions such as cell proliferation, endocytosis, and the response of cells to inflammatory and apoptotic stress signals (1–4).Sphingolipids are derived from the aliphatic amino alcohol sphingosine, which is formed from the precursors l-serine and palmitoyl-CoA. The condensation of serine with palmitoyl-CoA is catalyzed by the enzyme serine palmitoyltransferase (SPT)³ (EC 2.3.1.50) and leads to the intermediate 3-ketodihydrosphingosine. 3-Ketodihydrosphingosine is then rapidly converted to dihydrosphingosine (sphinganine) and dihydroceramide. The desaturation of dihydroceramide generates ceramide, and the breakdown of ceramide by ceramidase finally forms sphingosine. The sphingosine backbone of ceramide is usually O-linked to a polar head group such as phosphocholine or carbohydrates and amide-linked to an acyl group. The combination of the sphingosine backbone with different head groups, in particular with various oligosaccharides, leads to a complex variety of different sphingolipid metabolites (5, 6). Moreover, it was shown recently that SPT is also able to use l-alanine as an alternative substrate, thereby generating the atypical sphingoid base 1-deoxysphinganine (7).SPT belongs to the family of pyridoxal phosphate-dependent α-oxoamine synthases. Other members of this family include 5-aminolevulinic acid synthase, 2-amino-3 ketobutyrate ligase, and 8-amino-7-oxononanoate synthase (8). SPT is ubiquitously expressed, and enzyme activity has been detected in all tissues tested so far including brain, lung, liver, kidney, and muscle (9). SPT is essential for embryonic development, and homozygous SPT knock-out mice are not viable (10). SPT has been believed to be a heterodimer composed of two subunits, SPTLC1 and SPTLC2. The two subunits SPTLC1 and SPTLC2 show a similarity at AA level of ∼20% and are highly conserved among species. Although both subunits seem to be required for enzyme activity, only the SPTLC2 subunit contains a pyridoxal phosphate binding motif (8, 11).Recently, we identified and cloned a novel third SPT subunit (SPTLC3) (12). The SPTLC3 sequence shows 68% homology to the SPTLC2 subunit and also includes a pyridoxal phosphate consensus motive. The SPTLC3 gene is present in mammals, birds, and some lower vertebrates like fish (Danio rerio) and frog (Xenopus laevis) but not in invertebrate lineages. The SPTLC3 mRNA has been detected in most human tissues with a particularly high expression in placenta (12), indicating a special role for SPTLC3 during development and pregnancy. By using immunoprecipitation, native gel analysis, cross-linking studies, and size exclusion chromatography, it was demonstrated that the native SPT enzyme contains all three subunits and forms a protein complex with a molecular mass of about 460 kDa (13). However, because SPTLC2 and SPTLC3 are encoded by two distinct genes and expressed within the same cell types, we assume a distinct function for the two subunits. One of these differences might be altered substrate affinity or enzymatic activity. This issue is addressed in the present study.     

Phosphorylation of the Par Polarity Complex Protein Par3 at Serine 962 Is Mediated by Aurora A and Regulates Its Function in Neuronal Polarity

The Aurora kinases are a family of serine/threonine protein kinases that perform important functions during the cell cycle. Recently, it was shown that Drosophila Aurora A also regulates the asymmetric localization of Numb to the basal and the partitioning-defective (Par) complex to the apical cortex of neuroblasts by phosphorylating Par6. Here, we show that Aurora A is required for neuronal polarity. Suppression of Aurora A by RNA interference results in the loss of neuronal polarity. Aurora A interacts directly with the atypical protein kinase C binding domain of Par3 and phosphorylates it at serine 962. The phosphorylation of Par3 at serine 962 contributes to its function in the establishment of neuronal polarity.     

The Signaling Mechanisms Underlying Cell Polarity and Chemotaxis

Chemotaxis—the directed movement of cells in a gradient of chemoattractant—is essential for neutrophils to crawl to sites of inflammation and infection and for Dictyostelium discoideum (D. discoideum) to aggregate during morphogenesis. Chemoattractant-induced activation of spatially localized cellular signals causes cells to polarize and move toward the highest concentration of the chemoattractant. Extensive studies have been devoted to achieving a better understanding of the mechanism(s) used by a neutrophil to choose its direction of polarity and to crawl effectively in response to chemoattractant gradients. Recent technological advances are beginning to reveal many fascinating details of the intracellular signaling components that spatially direct the cytoskeleton of neutrophils and D. discoideum and the complementary mechanisms that make the cell''s front distinct from its back.Chemotaxis—the directed movement of cells in a gradient of chemoattractant—allows leukocytes to seek out sites of inflammation and infection, amoebas of Dictyostelium discoideum (D. discoideum) to aggregate, neurons to send projections to specific regions of the brain to find their synaptic partners, yeast cells to mate, and fibroblasts to move into the wound space (Fig. 1). In each case, chemoattractant-induced activation of spatially localized cellular signals causes cells to polarize and move toward the highest concentration of the chemoattractant. During chemotaxis, filamentous actin (F-actin) is polymerized asymmetrically at the upgradient edge of the cell (leading edge), providing the necessary force to thrust projections of the plasma membrane in the proper direction (see Mullins 2009). Neutrophilic leukocytes (neutrophils), for instance, can polarize and move up very shallow gradients, with a chemoattractant concentration ∼2% higher at the front than the back (Fig. 2) (Devreotes and Zigmond 1988). To restrict actin polymerization to the leading edge in such a shallow gradient, neutrophils must create a much steeper internal gradient of regulatory signals. In addition, distinctive actin–myosin contractile complexes are also formed at the sides and back of the cells (Fig. 2). The ability to create such distinctive segregation of actin assemblies enables neutrophils to move nearly 50 times more quickly than fibroblasts. The polarization response is self-organizing, which occurs even when the attractant concentration is uniform and apparently stimulating all portions of the plasma membrane at the same intensity; in the absence of a gradient, the direction of polarity is random, but all cells can be induced to polarize (Fig. 2). Thus, neutrophil polarization to chemoattractant stimulation represents a striking example of symmetry breaking from an unpolarized state to a polarized one.Open in a separate windowFigure 1.Examples of chemotaxis. (A) A human neutrophil chasing a Staphylococcus aureus microorganism on a blood film among red blood cells, notable for their dark color and principally spherical shape (imaged by David Rogers, courtesy of Thomas P. Stossel). Bar, 10 µm. Chemotaxis is also necessary for (B) D. discoideum to form multicellular aggregates during development (courtesy of M.J. Grimson and R.L. Blanton, Texas Tech University), and (C) for axons to find their way in the developing nervous system. Photo provided by Kathryn Tosney, University of Miami.Open in a separate windowFigure 2.(A–D) Polarization of a neutrophil in response to gradient of chemoattractant. Nomarski images of unpolarized neutrophil responding to a micropipette containing the chemoattractant fMLP (white circle) at (A) 5 s, (B) 30 s, (C) 81 s, and (D) 129 s of stimulation. Bar = 5 µm. (Figure is taken from Weiner et al. 1999, with permission.) Human neutrophils stimulated with fMLP show highly polarized morphology and asymmetric cytoskeletal assemblies. (E–G) Human neutrophils were stimulated by a uniform concentration of fMLP (100 nM) and fixed 2 min after stimulations. Fixed cells were stained for F-actin with rhodamine-phalloidin (E, red) and an antibody raised against activated myosin II (phosphorylated specifically at Ser19, p[19]-MLC) (F, green). These fluorescent images are merged with Nomarski image in (G). Bars, 10 µm.To enter an infected tissue, neutrophils require chemoattractants produced by host cells and microorganisms to migrate to the sites and infection and inflammation. Neutrophil chemotaxis also contributes to many inflammatory and autoimmune diseases, including rheumatoid arthritis, ischemia-reperfusion syndrome, acute respiratory distress, and systemic inflammatory response syndromes. Although the critical physiological functions of neutrophils have made their chemoattractants and chemoattractant receptors targets of intense investigation, understanding of the neutrophil polarity and directional migration has until recently lagged behind that of other cells. Over the past decade, experimentation with knockout mice and human neutrophil cell lines has begun to shed light on the complex intracellular signals responsible for neutrophil polarity. In this article, I summarize recent advances in the study of chemotactic signals in neutrophils, with some of the discussion also devoted to a related model—chemotaxis of D. discoideum. These soil amoebas grow as single cells, but on starvation chemotax into multicellular aggregates in response to secreted chemoattractants such as adenosine 3′,5′-monophosphate (cAMP).     

Establishment and Maintenance of Cell Polarity During Leukocyte Chemotaxis

The term polarity refers to the differential distribution of the macromolecular elements of a cell, resulting in its asymmetry in function, shape and/or content. Polarity is a fundamental property of all metazoan cells in at least some stages, and is pivotal to processes such as epithelial differentiation (apical/basal polarity), coordinated cell activity within the plane of a tissue (planar cell polarity), asymmetric cell division, and cell migration. In the last case, an apparently symmetric cell responds to directional cues provided by chemoattractants, creating a polarity axis that runs from the cell anterior, or leading edge, in which actin polymerization takes place, to the cell posterior (termed uropod in leukocytes), in which acto-myosin contraction occurs. Here we will review some of the molecular mechansisms through which chemoattractants break cell symmetry to trigger directed migration, focusing on cells of the immune system. We briefly highlight some common or apparently contradictory pathways reported as important for polarity in other cells, as this suggests conserved or cell type-specific mechanisms in eukaryotic cell chemotaxis.     

Establishment and Maintenance of Cell Polarity During Leukocyte Chemotaxis

The term polarity refers to the differential distribution of the macromolecular elements of a cell, resulting in its asymmetry in function, shape and/or content. Polarity is a fundamental property of all metazoan cells in at least some stages, and is pivotal to processes such as epithelial differentiation (apical/basal polarity), coordinated cell activity within the plane of a tissue (planar cell polarity), asymmetric cell division, and cell migration. In the last case, an apparently symmetric cell responds to directional cues provided by chemoattractants, creating a polarity axis that runs from the cell anterior, or leading edge, in which actin polymerization takes place, to the cell posterior (termed uropod in leukocytes), in which acto-myosin contraction occurs. Here we will review some of the molecular mechanisms through which chemoattractants break cell symmetry to trigger directed migration, focusing on cells of the immune system. We briefly highlight some common or apparently contradictory pathways reported as important for polarity in other cells, as this suggests conserved or cell type-specific mechanisms in eukaryotic cell chemotaxis.Key Words: chemotaxis, polarization, lipid rafts, signaling, cytoskeleton     

Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the (1248)SFYYS(1252) motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling.     

The aPKC/Par3/Par6 Polarity Complex and Membrane Order Are Functionally Interdependent in Epithelia During Vertebrate Organogenesis

The differential distribution of lipids between apical and basolateral membranes is necessary for many epithelial cell functions, but how this characteristic membrane organization is integrated within the polarity network during ductal organ development is poorly understood. Here we quantified membrane order in the gut, kidney and liver ductal epithelia in zebrafish larvae at 3–11 days post fertilization (dpf) with Laurdan 2‐photon microscopy. We then applied a combination of Laurdan imaging, antisense knock‐down and analysis of polarity markers to understand the relationship between membrane order and apical‐basal polarity. We found a reciprocal relationship between membrane order and the cell polarity network. Reducing membrane condensation by exogenously added oxysterol or depletion of cholesterol reduced apical targeting of the polarity protein, aPKC. Conversely, using morpholino knock down in zebrafish, we found that membrane order was dependent upon the Crb3 and Par3 polarity protein expression in ductal epithelia. Hence our data suggest that the biophysical property of membrane lipid packing is a regulatory element in apical basal polarity.     

Serine 123 Phosphorylation Modulates p21 Protein Stability and Activity by Suppressing Ubiquitin-independent Proteasomal Degradation

The cyclin-dependent kinase inhibitor p21^Waf1/Cip1 is a major regulator of the cell cycle and plays an important role in many cellular processes, including differentiation, stress response, apoptosis, and tumorigenesis. We previously cloned the gene encoding dog p21 and found that unlike its human ortholog, dog p21 is expressed as two isoforms, one high molecular mass band of 23 kDa and one low molecular mass band of 19 kDa. In the current study, we found that the high molecular mass band is phosphorylated, whereas the low molecular mass band is hypophosphorylated. Moreover, by generating multiple mutants of dog p21, we found that serine 123 and proline 124, which form a consensus site for proline-directed phosphorylation, are required for expression of the high molecular mass p21 isoform through phosphorylation at serine 123. Most importantly, we showed that serine 123 phosphorylation inhibits ubiquitin-independent proteasomal degradation of p21 protein and subsequently, prolongs p21 protein half-life and enhances the ability of p21 to suppress cell proliferation. Taken together, these data reveal that serine 123 phosphorylation modulates p21 protein stability and activity by suppressing ubiquitin-independent proteasomal degradation.     

Kidins220/ARMS Modulates the Activity of Microtubule-regulating Proteins and Controls Neuronal Polarity and Development

In order for neurons to perform their function, they must establish a highly polarized morphology characterized, in most of the cases, by a single axon and multiple dendrites. Herein we find that the evolutionarily conserved protein Kidins220 (kinase D-interacting substrate of 220-kDa), also known as ARMS (ankyrin repeat-rich membrane spanning), a downstream effector of protein kinase D and neurotrophin and ephrin receptors, regulates the establishment of neuronal polarity and development of dendrites. Kidins220/ARMS gain and loss of function experiments render severe phenotypic changes in the processes extended by hippocampal neurons in culture. Although Kidins220/ARMS early overexpression hinders neuronal development, its down-regulation by RNA interference results in the appearance of multiple longer axon-like extensions as well as aberrant dendritic arbors. We also find that Kidins220/ARMS interacts with tubulin and microtubule-regulating molecules whose role in neuronal morphogenesis is well established (microtubule-associated proteins 1b, 1a, and 2 and two members of the stathmin family). Importantly, neurons where Kidins220/ARMS has been knocked down register changes in the phosphorylation activity of MAP1b and stathmins. Altogether, our results indicate that Kidins220/ARMS is a key modulator of the activity of microtubule-regulating proteins known to actively regulate neuronal morphogenesis and suggest a mechanism by which it contributes to control neuronal development.     

PIM1 Kinase Phosphorylates the Human Transcription Factor FOXP3 at Serine 422 to Negatively Regulate Its Activity under Inflammation

Previous reports have suggested that human CD4⁺ CD25^hiFOXP3⁺ T regulatory cells (Tregs) have functional plasticity and may differentiate into effector T cells under inflammation. The molecular mechanisms underlying these findings remain unclear. Here we identified the residue serine 422 of human FOXP3 as a phosphorylation site that regulates its function, which is not present in murine Foxp3. PIM1 kinase, which is highly expressed in human Tregs, was found to be able to interact with and to phosphorylate human FOXP3 at serine 422. T cell receptor (TCR) signaling inhibits PIM1 induction, whereas IL-6 promotes PIM1 expression in in vitro expanded human Tregs. PIM1 negatively regulates FOXP3 chromatin binding activity by specifically phosphorylating FOXP3 at Ser⁴²². Our data also suggest that phosphorylation of FOXP3 at the Ser⁴¹⁸ site could prevent FOXP3 phosphorylation at Ser⁴²² mediated by PIM1. Knockdown of PIM1 in in vitro expanded human Tregs promoted FOXP3-induced target gene expression, including CD25, CTLA4, and glucocorticoid-induced tumor necrosis factor receptor (GITR), or weakened FOXP3-suppressed IL-2 gene expression and enhanced the immunosuppressive activity of Tregs. Furthermore, PIM1-specific inhibitor boosted FOXP3 DNA binding activity in in vitro expanded primary Tregs and also enhanced their suppressive activity toward the proliferation of T effector cells. Taken together, our findings suggest that PIM1 could be a new potential therapeutic target in the prevention and treatment of human-specific autoimmune diseases because of its ability to modulate the immunosuppressive activity of human Tregs.     

Polarity protein Par3 controls B-cell receptor dynamics and antigen extraction at the immune synapse

B-cell receptor (BCR) engagement with surface-tethered antigens leads to the formation of an immune synapse, which facilitates antigen uptake for presentation to T-lymphocytes. Antigen internalization and processing rely on the early dynein-dependent transport of BCR–antigen microclusters to the synapse center, as well as on the later polarization of the microtubule-organizing center (MTOC). MTOC repositioning allows the release of proteases and the delivery of MHC class II molecules at the synapse. Whether and how these events are coordinated have not been addressed. Here we show that the ancestral polarity protein Par3 promotes BCR–antigen microcluster gathering, as well as MTOC polarization and lysosome exocytosis, at the synapse by facilitating local dynein recruitment. Par3 is also required for antigen presentation to T-lymphocytes. Par3 therefore emerges as a key molecule in the coupling of the early and late events needed for efficient extraction and processing of immobilized antigen by B-cells.     

LIM and SH3 Protein -1 Modulates CXCR2-Mediated Cell Migration

Background

The chemokine receptor CXCR2 plays a pivotal role in migration of neutrophils, macrophages and endothelial cells, modulating several biological responses such as angiogenesis, wound healing and acute inflammation. CXCR2 is also involved in pathogenesis of chronic inflammation, sepsis and atherosclerosis. The ability of CXCR2 to associate with a variety of proteins dynamically is responsible for its effects on directed cell migration or chemotaxis. The dynamic network of such CXCR2 binding proteins is termed as “CXCR2 chemosynapse”. Proteomic analysis of proteins that co-immunoprecipitated with CXCR2 in neutrophil-like dHL-60 cells revealed a novel protein, LIM and SH3 protein 1 (LASP-1), binds CXCR2 under both basal and ligand activated conditions. LASP-1 is an actin binding cytoskeletal protein, involved in the cell migration.

Methodology/Principal Findings

We demonstrate that CXCR2 and LASP-1 co-immunoprecipitate and co-localize at the leading edge of migrating cells. The LIM domain of LASP-1 directly binds to the carboxy-terminal domain (CTD) of CXCR2. Moreover, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4. Using a site-directed and deletion mutagenesis approach, Iso323-Leu324 of the conserved LKIL motif on CXCR2-CTD was identified as the binding site for LASP-1. Interruption of the interaction between CXCR2-CTD and LIM domain of LASP-1 by dominant negative and knock down approaches inhibited CXCR2-mediated chemotaxis. Analysis for the mechanism for inhibition of CXCR2-mediated chemotaxis indicated that LASP-1/CXCR2 interaction is essential for cell motility and focal adhesion turnover involving activation of Src, paxillin, PAK1, p130CAS and ERK1/2.

Conclusions/Significance

We demonstrate here for the first time that LASP-1 is a key component of the “CXCR2 chemosynapse” and LASP-1 interaction with CXCR2 is critical for CXCR2-mediated chemotaxis. Furthermore, LASP-1 also directly binds the CTD of CXCR1, CXCR3 and CXCR4, suggesting that LASP-1 is a general mediator of CXC chemokine mediated chemotaxis. Thus, LASP-1 may serve as a new link coordinating the flow of information between chemokine receptors and nascent focal adhesions, especially at the leading edge. Thus the association between the chemokine receptors and LASP-1 suggests to the presence of a CXC chemokine receptor-LASP-1 pro-migratory module in cells governing the cell migration.     

Ustilago maydis Rho1 and 14-3-3 Homologues Participate in Pathways Controlling Cell Separation and Cell Polarity

Proteins of the 14-3-3 and Rho-GTPase families are functionally conserved eukaryotic proteins that participate in many important cellular processes such as signal transduction, cell cycle regulation, malignant transformation, stress response, and apoptosis. However, the exact role(s) of these proteins in these processes is not entirely understood. Using the fungal maize pathogen, Ustilago maydis, we were able to demonstrate a functional connection between Pdc1 and Rho1, the U. maydis homologues of 14-3-3ɛ and Rho1, respectively. Our experiments suggest that Pdc1 regulates viability, cytokinesis, chromosome condensation, and vacuole formation. Similarly, U. maydis Rho1 is also involved in these three essential processes and exerts an additional function during mating and filamentation. Intriguingly, yeast two-hybrid and epistasis experiments suggest that both Pdc1 and Rho1 could be constituents of the same regulatory cascade(s) controlling cell growth and filamentation in U. maydis. Overexpression of rho1 ameliorated the defects of cells depleted for Pdc1. Furthermore, we found that another small G protein, Rac1, was a suppressor of lethality for both Pdc1 and Rho1. In addition, deletion of cla4, encoding a Rac1 effector kinase, could also rescue cells with Pdc1 depleted. Inferring from these data, we propose a model for Rho1 and Pdc1 functions in U. maydis.Morphological switching is a unique attribute of all dimorphic fungi, which alternate between budding and filamentous growth. In some cases, as with mating, this is a prerequisite for genetic diversity for this subfamily of fungi. In addition, many dimorphic fungal pathogens rely on this ability in order to effectively invade their host. In general, the transition between these alternate life forms means a complete turnover of cellular and proteomic components, which often involves cell cycle arrest and/or cytoskeletal rearrangement. Although the cellular proteomes associated with these two processes share many components, there are both temporal and spatial regulations that are manifested during the transitional phase (4).Temporal-spatial regulation of the proteome during the dimorphic transition requires cooperation and synchronized communication among different regulatory pathways. Two highly intricate, yet well-established, signaling cascades that regulate fungal morphogenesis are the mitogen-activated protein kinase (MAPK) (34, 46) and protein kinase A pathways (11). These signaling cascades detect and perpetuate extracellular stimuli, e.g., pheromones and nutrients, which lead to phase transitions in dimorphic fungi. Although the mechanisms are not as fully understood, members of two highly conserved families of proteins, Rho/Rac GTPases and 14-3-3 proteins, have also been shown to control filamentation. Constituents of the Rho/Rac protein family have been shown to regulate actin organization (26, 35, 36), cytokinesis (3, 49, 52), cell integrity (42, 56), pathogenicity (29), signal transduction (22, 44, 56), and cell migration (8). Their activity is dependent upon the reversible binding of guanine nucleotides catalyzed by guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs) (15, 22, 23, 25, 44). Upon activation, Rho-GTPases stimulate downstream effector proteins such as p21-activated kinases (PAKs) (29, 51) or Rho kinase (ROCK) (36). Based on in silico analysis of the genome sequence, the fungal pathogen of maize, Ustilago maydis, contains six different Rho/Rac encoding genes: cdc42, rac1, and four additional genes predicted to encode Rho-like proteins (20). Of these, only the roles of Cdc42 and Rac1 have been examined in depth. Cdc42 was shown to regulate cytokinesis, while Rac1 regulates hyphal development in U. maydis (26). We examine here the place of a Rho1 homologue, Rho1, in U. maydis cell morphology, polarity, and development.Similarly, the highly conserved, ubiquitously expressed 14-3-3 proteins that are found in most eukaryotes have also been shown to contribute to cellular differentiation and cytoskeletal organization. Like Rho-GTPases, 14-3-3 proteins play multiple roles, in cytoskeletal function, cell cycle regulation, apoptosis, and the regulation of a variety of signaling pathways (17, 19, 33, 50). These acidic proteins have been found in each cellular compartment and most organisms examined possess multiple isoforms: seven isoforms are found in mammals, and as many as fifteen isoforms have been identified in plants (31). Interestingly, the yeast Saccharomyces cerevisiae, the fruit fly, Drosophila melanogaster, and the nematode, Caenorhabditis elegans, each possess only two 14-3-3 isoforms (50), while Candida albicans contains a single isoform (37). They function typically by binding their particular ligands at phosphoserine or phosphothreonine residues (50). It is not clear what 14-3-3 proteins do in the processes mentioned above, whether they act as scaffolds or effectors. Inspection of the U. maydis genome sequence revealed that this organism could be ideal for the study of 14-3-3 proteins because, unlike most other organisms, the U. maydis genome contains only a single 14-3-3 homologue. Due to its predicted binding of phosphorylated proteins, we named this homologue Pdc1 (for phosphorylation domain coupling protein [10]). Recently, the protein (also designated Bmh1 [32]) was also shown to be involved in cell cycle regulation.Despite the functional differences between Rho-GTPases and 14-3-3 proteins, we provide evidence that members of these two families participate in the same regulatory cascade(s) that control morphogenesis in the dimorphic fungus U. maydis. We are able to demonstrate that both Pdc1 and Rho1 are essential for cell viability. In addition, overexpression of Rho1 led to the reduction of filamentation. Overexpression of Rac1 triggers filamentation in U. maydis (13, 29). We show here that deleting Rac1 eliminates the lethal effect imposed by either Rho1 or Pdc1 depletion. Our results have led us to predict that both Rho1 and Pdc1 are negative regulators of Rac1 in U. maydis and that they play important roles in polarized growth and cytokinesis.     

Nectin-1 Binds and Signals through the Fibroblast Growth Factor Receptor

Nectins belong to a family of immunoglobulin (Ig)-like cell-adhesion molecules comprising four members, nectin-1 through nectin-4. Nectins are involved in formation of the mechanical adhesive puncta adherentia junctions of synapses. Nectins share the same overall structural topology with an extracellular region containing three Ig modules, a transmembrane region, and a cytoplasmic region. In nectin-1, the first and second Ig module in the extracellular region are necessary for the trans-interaction with nectin-3 and formation of cis-dimers, respectively. The function of the third Ig module of nectin-1 remains unknown. We here report the structure in solution of the third, membrane-proximal Ig module of mouse nectin-1 (nectin-1 Ig3) solved by means of nuclear magnetic resonance (NMR) spectroscopy. It belongs to the C1 set of the Ig superfamily. Nectin-1 Ig3 was produced as a recombinant protein and induced neurite outgrowth in primary cultures of hippocampal and cerebellar granule neurons, an effect abolished by treatment with the fibroblast growth factor receptor (FGFR) inhibitor SU5402, or by transfection with a dominant-negative FGFR1 construct. We showed by surface plasmon resonance (SPR) analysis that nectin-1 Ig3 directly interacted with various isoforms of FGFR. Nectin-1 Ig3 induced phosphorylation of FGFR1c in the same manner as the whole nectin-1 ectodomain, and promoted survival of cerebellar granule neurons induced to undergo apoptosis. Finally, we constructed a peptide, nectide, by employing in silico modeling of various FGFR ligand-binding sites. Nectide mimicked all the effects of nectin-1 Ig3. We suggest that FGFR is a downstream signaling partner of nectin-1.     

Cell Survival and Polarity of Drosophila Follicle Cells Require the Activity of Ecdysone Receptor B1 Isoform

Proper assembly and maintenance of epithelia are critical for normal development and homeostasis. Here, using the Drosophila ovary as a model, we identify a role for the B1 isoform of the ecdysone receptor (EcR-B1) in this process. We performed a reverse genetic analysis of EcR-B1 function during oogenesis and demonstrate that silencing of this receptor isoform causes loss of integrity and multilayering of the follicular epithelium. We show that multilayered follicle cells lack proper cell polarity with altered distribution of apical and basolateral cell polarity markers including atypical-protein kinase C (aPKC), Discs-large (Dlg), and Scribble (Scrib) and aberrant accumulation of adherens junctions and F-actin cytoskeleton. We find that the EcR-B1 isoform is required for proper follicle cell polarity both during early stages of oogenesis, when follicle cells undergo the mitotic cell cycle, and at midoogenesis when these cells stop dividing and undergo several endocycles. In addition, we show that the EcR-B1 isoform is required during early oogenesis for follicle cell survival and that disruption of its function causes apoptotic cell death induced by caspase.     

AMPK/LKB1 Signaling in Epithelial Cell Polarity and Cell Division

Cells must coordinate diverse processes including cell division, cell migration, and cell polarity with the cell’s metabolic status. How single molecules coordinate these seemingly distinct cell biological events remains relatively unexplored. AMP-activated protein kinase (AMPK) sits at a unique position as a proposed energy sensor that can interface with diverse signaling molecules ranging from LKB1 to mammalian target of rapamycin (mTOR), affecting processes from ribosomal biogenesis to actin regulation. Determining biologically relevant direct kinase targets remains challenging. Alternatively, one can genetically inactivate a kinase and subsequently characterize cellular and whole animal phenotypes without the kinase’s activity. Recent genetic studies inactivating AMPK activity in Drosophila indicate unanticipated roles for AMPK as a regulator of epithelial polarity, consistent with known roles of an upstream activator, LKB1 as a PAR (partioning defective) mutant in Caenorhabditis elegans and polarity regulator. Additional genetic analyses demonstrate that both AMPK and LKB1 function are required for faithful chromosomal segregation during mitosis. At least some of these apparently divergent phenotypes may be mediated through myosin regulatory light chain, and presumably the acto-myosin complex, which can affect both polarity and cell division. Chromosomal integrity defects could also be consistent with LKB1’s role as a known human tumor suppressor gene. Elucidating the molecular players that interface with AMPK and their potential energy dependent regulation remains an important challenge to fully understand AMPK signaling.