Nonsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine comprising the genomic RNA completely encapsidated by the viral nucleocapsid protein and associated with the viral polymerase. The catalytic core of this protein-RNA complex is a 250-kDa multifunctional large (L) polymerase protein that contains enzymatic activities for nucleotide polymerization as well as for each step of mRNA cap formation. Working with vesicular stomatitis virus (VSV), a prototype of NNS RNA viruses, we used negative stain electron microscopy (EM) to obtain a molecular view of L, alone and in complex with the viral phosphoprotein (P) cofactor. EM analysis, combined with proteolytic digestion and deletion mapping, revealed the organization of L into a ring domain containing the RNA polymerase and an appendage of three globular domains containing the cap-forming activities. The capping enzyme maps to a globular domain, which is juxtaposed to the ring, and the cap methyltransferase maps to a more distal and flexibly connected globule. Upon P binding, L undergoes a significant rearrangement that may reflect an optimal positioning of its functional domains for transcription. The structural map of L provides new insights into the interrelationship of its various domains, and their rearrangement on P binding that is likely important for RNA synthesis. Because the arrangement of conserved regions involved in catalysis is homologous, the structural insights obtained for VSV L likely extend to all NNS RNA viruses.L protein | structure and function | viral replication N onsegmented negative-strand (NNS) RNA viruses initiate infection by delivering into the host cell a highly specialized RNA synthesis machine. This machine consists of a ribonucleoprotein complex (RNP) comprising the genomic RNA completely coated by the viral nucleocapsid (N) protein and associated with the RNA-dependent RNA polymerase (RdRP) complex (1). The catalytic core of the RNP is a single large (L) 250-kDa protein that contains enzymatic activities for nucleotide polymerization, mRNA cap addition, cap methylation, and polyadenylation (2-6). The location of all of the enzymatic activities necessary for transcription within a single polypeptide chain contrasts with the arrangement exhibited by the host cell and many other viruses, in which the different activities reside within separate proteins that assemble into a larger transcription complex (7).Our understanding of the different activities of NNS RNA virus L proteins has been largely shaped by studies of vesicular stomatitis virus (VSV) because it is the only member of this order of viruses for which robust transcription can be reconstituted in vitro. The enzymatic activities of VSV L have been mapped at the single amino acid level. Within the primary sequence of L are six conserved regions (CRs I-VI) shared among all NNS RNA virus L proteins (8). The RdRP activity maps to CRIII (3), and it is also required for polyadenylation, which occur...

Rift Valley fever virus (RVFV), a Phlebovirus with a genome consisting of three single-stranded RNA segments, is spread by infected mosquitoes and causes large viral outbreaks in Africa. RVFV encodes a nucleoprotein (N) that encapsidates the viral RNA. The N protein is the major component of the ribonucleoprotein complex and is also required for genomic RNA replication and transcription by the viral polymerase. Here we present the 1.6 Å crystal structure of the RVFV N protein in hexameric form. The ring-shaped hexamers form a functional RNA binding site, as assessed by mutagenesis experiments. Electron microscopy (EM) demonstrates that N in complex with RNA also forms rings in solution, and a single-particle EM reconstruction of a hexameric N-RNA complex is consistent with the crystallographic N hexamers. The ring-like organization of the hexamers in the crystal is stabilized by circular interactions of the N terminus of RVFV N, which forms an extended arm that binds to a hydrophobic pocket in the core domain of an adjacent subunit. The conformation of the N-terminal arm differs from that seen in a previous crystal structure of RVFV, in which it was bound to the hydrophobic pocket in its own core domain. The switch from an intra- to an inter-molecular interaction mode of the N-terminal arm may be a general principle that underlies multimerization and RNA encapsidation by N proteins from Bunyaviridae. Furthermore, slight structural adjustments of the N-terminal arm would allow RVFV N to form smaller or larger ring-shaped oligomers and potentially even a multimer with a super-helical subunit arrangement. Thus, the interaction mode between subunits seen in the crystal structure would allow the formation of filamentous ribonucleocapsids in vivo. Both the RNA binding cleft and the multimerization site of the N protein are promising targets for the development of antiviral drugs.

Netrin-1 acts as a chemoattractant molecule to guide commissural neurons (CN) toward the floor plate by interacting with the receptor deleted in colorectal cancer (DCC). The molecular mechanisms underlying Netrin-1–DCC signaling are still poorly characterized. Here, we show that DCC is phosphorylated in vivo on tyrosine residues in response to Netrin-1 stimulation of CN and that the Src family kinase inhibitors PP2 and SU6656 block both Netrin-1–dependent phosphorylation of DCC and axon outgrowth. PP2 also blocks the reorientation of Xenopus laevis retinal ganglion cells that occurs in response to Netrin-1, which suggests an essential role of the Src kinases in Netrin-1–dependent orientation. Fyn, but not Src, is able to phosphorylate the intracellular domain of DCC in vitro, and we demonstrate that Y1418 is crucial for DCC axon outgrowth function. Both DCC phosphorylation and Netrin-1–induced axon outgrowth are impaired in Fyn−/− CN and spinal cord explants. We propose that DCC is regulated by tyrosine phosphorylation and that Fyn is essential for the response of axons to Netrin-1.

The Rho GTPases RhoA, Rac1, and Cdc42 play a major role in regulating the reorganization of the actin cytoskeleton. We recently identified CdGAP, a novel GTPase-activating protein with activity toward Rac1 and Cdc42. CdGAP consists of a N-terminal GAP domain, a central domain, and a C-terminal proline-rich domain. Here we show that through a subset of its Src homology 3 domains, the endocytic protein intersectin interacts with CdGAP. In platelet-derived growth factor-stimulated Swiss 3T3 cells, intersectin co-localizes with CdGAP and inhibits its GAP activity toward Rac1. Intersectin-Src homology 3 also inhibits CdGAP activity in GAP assays in vitro. Although the C-terminal prolinerich domain of CdGAP is required for the regulation of its GAP activity by intersectin both in vivo and in vitro, it is not necessary for CdGAP-intersectin interaction. Our data suggest that the central domain of CdGAP is required for CdGAP-intersectin interaction. Thus, we propose a model in which intersectin binding results in a change of CdGAP conformation involving the prolinerich domain that leads to the inhibition of its GAP activity. These observations provide the first demonstration of a direct regulation of RhoGAP activity through a protein-protein interaction and suggest a function for intersectin in Rac1 regulation and actin dynamics.

Rho GTPases regulate multiple cellular processes affecting both cell proliferation and cytoskeletal dynamics. Their cycling between inactive GDP-and active GTP-bound states is tightly regulated by guanine nucleotide exchange factors and GTPase-activating proteins (GAPs). We have previously identified CdGAP (for Cdc42 GTPase-activating protein) as a specific GAP for Rac1 and Cdc42. CdGAP consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP is a member of the impressively large number of mammalian RhoGAP proteins that is well conserved among both vertebrates and invertebrates. In mice, we find two predominant isoforms of CdGAP differentially expressed in specific tissues. We report here that CdGAP is highly phosphorylated in vivo on serine and threonine residues. We find that CdGAP is phosphorylated downstream of the MEK-extracellular signal-regulated kinase (ERK) pathway in response to serum or platelet-derived growth factor stimulation. Furthermore, CdGAP interacts with and is phosphorylated by ERK-1 and RSK-1 in vitro. A putative DEF (docking for ERK FXFP) domain located in the proline-rich region of CdGAP is required for efficient binding and phosphorylation by ERK1/2. We identify Thr 776 as an in vivo target site of ERK1/2 and as an important regulatory site of CdGAP activity. Together, these data suggest that CdGAP is a novel substrate of ERK1/2 and mediates cross talk between the Ras/ mitogen-activated protein kinase pathway and regulation of Rac1 activity.

Background information. Rho GTPases regulate a wide range of cellular functions affecting both cell proliferation and cytoskeletal dynamics. They cycle between inactive GDP-and active GTP-bound states. This cycle is tightly regulated by GEFs (guanine nucleotide-exchange factors) and GAPs (GTPase-activating proteins). Mouse CdGAP (mCdc42 GTPase-activating protein) has been previously identified and characterized as a specific GAP for Rac1 and Cdc42, but not for RhoA. It consists of an N-terminal RhoGAP domain and a C-terminal proline-rich region. In addition, CdGAP-related genes are present in both vertebrates and invertebrates. We have recently reported that two predominant isoforms of CdGAP (250 and 90 kDa) exist in specific mouse tissues.Results. In the present study, we have identified and characterized human CdGAP (KIAA1204) which shares 76% sequence identity to the long isoform of mCdGAP (mCdGAP-l). Similar to mCdGAP, it is active in vitro and in vivo on both Cdc42 and Rac1, but not RhoA, and is phosphorylated in vivo on serine and threonine residues. In contrast with mCdGAP-l, human CdGAP interacts with ERK1/2 (extracellular-signal-regulated kinase 1/2) through a region that does not involve a DEF (docking site for ERK Phe-Xaa-Phe-Pro) domain. Also, the tissue distribution of CdGAP proteins appears to be different between human and mouse species. Interestingly, we found that CdGAP proteins cause membrane blebbing in COS-7 cells. Conclusions.Our results suggest that CdGAP properties are well conserved between human and mouse species, and that CdGAP may play an unexpected role in apoptosis.

Rho GTPases regulate a multitude of cellular processes from cytoskeletal reorganization to gene transcription and are negatively regulated by GTPase-activating proteins (GAPs). Cdc42 GTPase-activating protein (CdGAP) is a ubiquitously expressed GAP for Rac1 and Cdc42. In this study, we set out to identify CdGAP-binding partners and, using a yeast two-hybrid approach, glycogen synthase kinase 3␣ (GSK-3␣) was identified as a partner for CdGAP. GSK-3 exists in two isoforms, ␣ and ␤, and is involved in regulating many cellular functions from insulin response to tumorigenesis. We show that GSK-3␣ and -␤ interact with CdGAP in mammalian cells. We also demonstrate that GSK-3 phosphorylates CdGAP both in vitro and in vivo on Thr-776, which we have previously shown to be an ERK 1/2 phosphorylation site involved in CdGAP regulation. We report that the mRNA and protein levels of CdGAP are increased upon serum stimulation and that GSK-3 activity is necessary for the up-regulation of the protein levels of CdGAP but not for the increase in mRNA. We conclude that GSK-3 is an important regulator of CdGAP and that regulation of CdGAP protein levels by serum presents a novel mechanism for cells to control Cdc42/Rac1 GTPase signaling pathways.The Rho subfamily of small GTPases controls a wide variety of cellular functions. RhoA, Rac1, and Cdc42 are the best known members of this family, and they are most often associated with their roles as regulators of cytoskeleton remodeling and as key mediators of the activation of transcription of genes downstream of growth factor receptors (1). Much evidence exists linking Rho GTPases to transformation of cells; however, contrary to the Ras gene, activating mutations in Rho genes are rarely found in human cancers (2, 3). It seems instead that the expression of Rho GTPases and expression and function of regulators of the Rho subfamily of GTPases are altered during cellular transformation (2, 3).Rho GTPases act in a cycle as molecular switches with an active GTP-bound form and an inactive GDP-bound form (1). The GTPase-activating proteins (GAPs) 5 negatively regulate the GTPases by enhancing the hydrolysis of GTP to GDP (1). To date, ϳ70 human genes are predicted to encode for potential RhoGAP proteins (4), which is roughly triple the number of Rho GTPases (1). This lends weight to the notion that regulators like the RhoGAPs tightly control Rho GTPases and lend context-dependent specificity to their processes. Thus, the activity of RhoGAPs must be highly regulated in both spatial and temporal fashions. RhoGAPs are regulated at the protein level by a variety of mechanisms ranging from protein-protein interactions to phosphorylation, lipid interactions, and proteolytic degradation (5).Cdc42 GTPase-activating protein (CdGAP) has been shown to regulate both Cdc42 and Rac1 in vitro and in vivo and exists in two main isoforms: a short form of 820 amino acids containing an N-terminal RhoGAP domain, a central region, and a C-terminal proline-rich region (PRD) (6, 7) and a long isoform comprising the...

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