A Small Conserved Domain in the Yeast Spa2p Is Necessary and Sufficient for Its Polarized Localization

SPA2 encodes a yeast protein that is one of the first proteins to localize to sites of polarized growth, such as the shmoo tip and the incipient bud. The dynamics and requirements for Spa2p localization in living cells are examined using Spa2p green fluorescent protein fusions. Spa2p localizes to one edge of unbudded cells and subsequently is observable in the bud tip. Finally, during cytokinesis Spa2p is present as a ring at the mother–daughter bud neck. The bud emergence mutants bem1 and bem2 and mutants defective in the septins do not affect Spa2p localization to the bud tip. Strikingly, a small domain of Spa2p comprised of 150 amino acids is necessary and sufficient for localization to sites of polarized growth. This localization domain and the amino terminus of Spa2p are essential for its function in mating. Searching the yeast genome database revealed a previously uncharacterized protein which we name, Sph1p (Spa2p homolog), with significant homology to the localization domain and amino terminus of Spa2p. This protein also localizes to sites of polarized growth in budding and mating cells. SPH1, which is similar to SPA2, is required for bipolar budding and plays a role in shmoo formation. Overexpression of either Spa2p or Sph1p can block the localization of either protein fused to green fluorescent protein, suggesting that both Spa2p and Sph1p bind to and are localized by the same component. The identification of a 150–amino acid domain necessary and sufficient for localization of Spa2p to sites of polarized growth and the existence of this domain in another yeast protein Sph1p suggest that the early localization of these proteins may be mediated by a receptor that recognizes this small domain.Polarized cell growth and division are essential cellular processes that play a crucial role in the development of eukaryotic organisms. Cell fate can be determined by cell asymmetry during cell division (Horvitz and Herskowitz, 1992; Cohen and Hyman, 1994; Rhyu and Knoblich, 1995). Consequently, the molecules involved in the generation and maintenance of cell asymmetry are important in the process of cell fate determination. Polarized growth can occur in response to external signals such as growth towards a nutrient (Rodriguez-Boulan and Nelson, 1989; Eaton and Simons, 1995) or hormone (Jackson and Hartwell, 1990a , b ; Segall, 1993; Keynes and Cook, 1995) and in response to internal signals as in Caenorhabditis elegans (Goldstein et al., 1993; Kimble, 1994; Priess, 1994) and Drosophila melanogaster (St Johnston and Nusslein-Volhard, 1992; Anderson, 1995) early development. Saccharomyces cerevisiae undergo polarized growth towards an external cue during mating and to an internal cue during budding. Polarization towards a mating partner (shmoo formation) and towards a new bud site requires a number of proteins (Chenevert, 1994; Chant, 1996; Drubin and Nelson, 1996). Many of these proteins are necessary for both processes and are localized to sites of polarized growth, identified by the insertion of new cell wall material (Tkacz and Lampen, 1972; Farkas et al., 1974; Lew and Reed, 1993) to the shmoo tip, bud tip, and mother–daughter bud neck. In yeast, proteins localized to growth sites include cytoskeletal proteins (Adams and Pringle, 1984; Kilmartin and Adams, 1984; Ford, S.K., and J.R. Pringle. 1986. Yeast. 2:S114; Drubin et al., 1988; Snyder, 1989; Snyder et al., 1991; Amatruda and Cooper, 1992; Lew and Reed, 1993; Waddle et al., 1996), neck filament components (septins) (Byers and Goetsch, 1976; Kim et al., 1991; Ford and Pringle, 1991; Haarer and Pringle, 1987; Longtine et al., 1996), motor proteins (Lillie and Brown, 1994), G-proteins (Ziman, 1993; Yamochi et al., 1994; Qadota et al., 1996), and two membrane proteins (Halme et al., 1996; Roemer et al., 1996; Qadota et al., 1996). Septins, actin, and actin-associated proteins localize early in the cell cycle, before a bud or shmoo tip is recognizable. How this group of proteins is localized to and maintained at sites of cell growth remains unclear.Spa2p is one of the first proteins involved in bud formation to localize to the incipient bud site before a bud is recognizable (Snyder, 1989; Snyder et al., 1991; Chant, 1996). Spa2p has been localized to where a new bud will form at approximately the same time as actin patches concentrate at this region (Snyder et al., 1991). An understanding of how Spa2p localizes to incipient bud sites will shed light on the very early stages of cell polarization. Later in the cell cycle, Spa2p is also found at the mother–daughter bud neck in cells undergoing cytokinesis. Spa2p, a nonessential protein, has been shown to be involved in bud site selection (Snyder, 1989; Zahner et al., 1996), shmoo formation (Gehrung and Snyder, 1990), and mating (Gehrung and Snyder, 1990; Chenevert et al., 1994; Yorihuzi and Ohsumi, 1994; Dorer et al., 1995). Genetic studies also suggest that Spa2p has a role in cytokinesis (Flescher et al., 1993), yet little is known about how this protein is localized to sites of polarized growth.We have used Spa2p green fluorescent protein (GFP)¹ fusions to investigate the early localization of Spa2p to sites of polarized growth in living cells. Our results demonstrate that a small domain of ∼150 amino acids of this large 1,466-residue protein is sufficient for targeting to sites of polarized growth and is necessary for Spa2p function. Furthermore, we have identified and characterized a novel yeast protein, Sph1p, which has homology to both the Spa2p amino terminus and the Spa2p localization domain. Sph1p localizes to similar regions of polarized growth and sph1 mutants have similar phenotypes as spa2 mutants.     

Arachidonic Acid Stimulates Cell Adhesion through a Novel p38 MAPK-RhoA Signaling Pathway That Involves Heat Shock Protein 27

Rho GTPases are critical components of cellular signal transduction pathways. Both hyperactivity and overexpression of these proteins have been observed in human cancers and have been implicated as important factors in metastasis. We previously showed that dietary n-6 fatty acids increase cancer cell adhesion to extracellular matrix proteins, such as type IV collagen. Here we report that in MDA-MB-435 human melanoma cells, arachidonic acid activates RhoA, and inhibition of RhoA signaling with either C3 exoenzyme or dominant negative Rho blocked arachidonic acid-induced cell adhesion. Inhibition of the Rho kinase (ROCK) with either small molecule inhibitors or ROCK II-specific small interfering RNA (siRNA) blocked the fatty acid-induced adhesion. However, unlike other systems, inhibition of ROCK did not block the activation of p38 mitogen-activated protein kinase (MAPK); instead, Rho activation depended on p38 MAPK activity and the presence of heat shock protein 27 (HSP27), which is phosphorylated downstream of p38 after arachidonic acid treatment. HSP27 associated with p115RhoGEF in fatty acid-treated cells, and this association was blocked when p38 was inhibited. Furthermore, siRNA knockdown of HSP27 blocked the fatty acid-stimulated Rho activity. Expression of dominant negative p115-RhoGEF or p115RhoGEF-specific siRNA inhibited both RhoA activation and adhesion on type IV collagen, whereas a constitutively active p115RhoGEF restored the arachidonic acid stimulation in cells in which the p38 MAPK had been inhibited. These data suggest that n-6 dietary fatty acids stimulate a set of interactions that regulates cell adhesion through RhoA and ROCK II via a p38 MAPK-dependent association of HSP27 and p115RhoGEF.The ability of tumor cells to metastasize to secondary sites is a hallmark of neoplastic disease. Unfortunately, this propensity to spread is the primary cause of morbidity and death in cancer patients (1). Metastasis is clearly a highly regulated, multistep process that occurs in a spatiotemporal manner (2–4). To escape the restrictive compartment boundaries characteristic of adult tissue, separate intravasation and extravasation steps requiring alterations in co-adhesion, adhesion, invasion, and migration must occur. Execution of these biological processes, involving multiple proteins and cellular organelles, require highly coordinated cell signaling mechanisms.The Rho family of small GTPases regulates many facets of cytoskeletal rearrangements that facilitate cell attachment and migration (5–7). Rho GTPases act as molecular switches by changing from an inactive GDP-bound conformation to an active GTP-bound conformation, thereby regulating a signaling pathway. These proteins are directly regulated by Rho guanine nucleotide exchange factors (GEFs),² Rho GTPase activating proteins, and Rho GDP-dissociation inhibitors (8–12). RhoGEFs bind to the GTPase to catalyze the dissociation of GDP, allowing the binding of GTP and thereby promoting Rho activation (8). The RGS (regulators of G protein signaling) domain-containing RhoGEFs are a recently described family of GEFs. Currently, there are three members of this family, PDZ-RhoGEF, LARG, and p115RhoGEF (13–15), in which the RGS domains function as a heterotrimeric GTPase-activating domain (13, 15, 16). The RGS family of RhoGEFs has been shown to regulate Rho during several processes including cytoskeletal rearrangements, cell adhesion, and cancer progression (17–21).There is significant interplay between the activity of small GTPases and signaling derived from fatty acid metabolism (22–28). Linoleic acid, which is metabolized to arachidonic acid, is an n-6 polyunsaturated fatty acid that is present at high levels in most western diets (29). In animal models, diets high in n-6 polyunsaturated fatty acids have been shown to enhance tumor progression and metastasis (30, 31). Additionally, arachidonic acid is stored in cell membranes and is made available by phospholipases under conditions of increased inflammatory response (32). Arachidonic acid is further metabolized by cyclooxygenases (COX), lipoxygenases (LOX), and cytochrome P450 monooxygenases to yield bioactive products that have myriad effects on cells, and altered metabolism of arachidonic acid by COX, LOX, and P450 has been implicated in cancer progression (31, 33–36).We have studied mechanisms of cell adhesion using the MDA-MB-435 cells as a model of a highly metastatic human cancer cell line (37). These cells have been extensively studied for their ability to recapitulate the metastatic cascade in vivo and in vitro, although recent work indicates that the cells currently in use are most likely a human melanoma line (38). We initially observed that arachidonic acid (AA) enhanced adhesion of MDA-MB-435 cells to type IV collagen through specific integrin-mediated pathways (37). Exogenous AA led to the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase 2 and the phosphorylation of heat shock protein 27 (HSP27) via a p38 MAPK-dependent process (39). Inhibition of p38 MAPK activation blocked cell adhesion as did function-blocking antibodies specific for subunits of the collagen receptor (40). More recently, we identified the key metabolite of AA (15-(S)- hydroxyeicosatetraenoic acid) and the upstream kinases (TAK1 and MKK6) that are responsible for activation of p38 MAPK in this system (41).In this study we investigated the role of Rho activation in the MDA-MB-435 cells after exposure to arachidonic acid. Several aspects of the regulation of Rho signaling in these cells provide insights into the cross-talk between important signaling pathways.     

Insect Virus Proteins (FALPE and p10) Self-Associate To Form Filaments in Infected Cells

Entomopoxviruses and baculoviruses are pathogens of insects which replicate in the cytoplasm and nuclei of their host cells, respectively. During the late stages of infection, both groups of viruses produce occlusion bodies which serve to protect virions from the external environment. Immunofluorescence and electron microscopy studies have shown that large bundles of filaments are associated with these occlusion bodies. Entomopoxviruses produce cytoplasmic fibrils which appear to be composed of the filament-associated late protein of entomopoxviruses (FALPE). Baculoviruses, on the other hand, yield filaments in the nuclei and cytoplasm of the infected cell which are composed of a protein called p10. Despite significant differences in their sequences, FALPE and p10 have similar hydrophilicity profiles, and each has a proline-rich stretch of amino acids at its carboxyl terminus. Evidence that FALPE and p10 could produce filaments in the absence of other viral proteins is presented. When FALPE was expressed in insect cells from a recombinant baculovirus, filaments similar to those produced by the wild-type Amsacta moorei entomopoxvirus were observed. In addition, when expression plasmids containing FALPE or p10 genes were transfected into Vero monkey kidney cells, filament structures similar to those found in infected insect cells were produced. The manner in which FALPE and p10 subunits interact to form polymers was investigated through deletion and site-specific mutagenesis in conjunction with immunofluorescence microscopy, yeast two-hybrid protein interaction analysis, and chemical cross-linking of adjacent molecules. These studies indicated that the amino termini of FALPE and p10 were essential for subunit interaction. Although deletion of the carboxy termini did not affect this interaction, it did inhibit filament formation. In addition, modification of several potential sites for phosphorylation also abolished filament assembly. We concluded that although the sequences of FALPE and p10 were different, the structural and functional properties of the two polypeptides appeared to be similar.Cytoskeletal elements have previously been demonstrated to be involved in several aspects of virus assembly (39, 66). For example, vaccinia virus has been shown to associate with actin during its release from the plasma membrane (15), while adenovirus is transported through the cytoplasm to the nucleus through its interaction with microtubules (17, 38). Actin has been implicated in the transport of baculovirus nucleocapsids to the nucleus (10). Other viruses contain actin in their envelopes along with viral surface glycoproteins, implying some role in the budding process (34, 54, 58). In addition, cytochalasin D, a disruptor of microfilaments, has been shown to impair the assembly of a number of different viruses (18, 42, 45). Most viruses use preexisting microtubule or microfilament proteins derived from host cells in these processes. However, we have recently demonstrated that insect poxviruses establish their own filament network during the later stages of infection, using a protein encoded by the viral genome (2).Entomopoxviruses (EPVs) are insect pathogens which replicate in the cytoplasm of infected cells and are members of the poxvirus family (reviewed in references 3 to 5 and 22). The genomes of these viruses consist of linear double-stranded DNA molecules which are 130 to 300 kb in length. Amsacta moorei EPV (AmEPV) can be grown in cultured insect cells and is the most studied member of this group of viruses (22–25, 27, 40, 50). AmEPV derives its name from the Indian red army worm, a larva from the Lepidoptera family and the host from which the virus was originally isolated (23, 25, 50). Baculoviruses also infect Lepidoptera larvae but instead replicate in the nuclei of their host cells (44). A number of baculoviruses have been studied, but knowledge of Autographa californica nuclear polyhedrosis virus (AcNPV), which infects a wide variety of larvae including that of the alfalfa leaf hopper, is most extensive (44). This virus is used routinely to produce recombinant proteins in insect virus expression systems (36, 44, 46, 49).A common property of EPVs and baculoviruses is the formation of large intracellular structures known as occlusion bodies which assemble during the late stages of viral infection. Virions are embedded within these occlusion bodies, and the process serves to protect the virus from the external environment. In the case of baculoviruses, the occlusion bodies are called polyhedra and are composed predominantly of a 31-kDa protein called polyhedrin (52). The occlusion bodies of EPVs are known as spheroids and consist mainly of a 110-kDa protein known as spheroidin (6, 9, 27, 55). Spheroidin and polyhedrin do not appear to exhibit sequence homology (6, 27, 52). A multilamellar envelope also appears to surround both polyhedra and spheroids and may help to stabilize these structures during assembly (2, 53).During the late phases of AmEPV and baculovirus infections, large bundles of filaments also appear to accumulate in the infected insect cells. In the case of AmEPV, these structures are present in the cytoplasm (2, 22, 23, 40), while those found in cells infected with baculoviruses reside both in the cytoplasm and in the nucleus (1, 14, 57). Baculovirus fibrils are composed primarily of a 10-kDa protein called p10 (47, 59). The p10 gene sequences from AcNPV, Orgyia pseudotsugata nuclear polyhedrosis virus (OpNPV), Bombyx mori nuclear polyhedrosis virus, Perina nuda nuclear polyhedrosis virus, Spodoptera exigua nuclear polyhedrosis virus (SeNPV), and Choristoneura fumiferana nuclear polyhedrosis virus (CfNPV) have been reported (13, 32, 35, 66–68). Although the different p10 protein sequences only exhibit 39 to 51% identity and molecules from different species cannot interact with one another, it is believed that the polypeptides must be structurally and functionally similar (61, 66). Deletion mutagenesis of AcNPV p10 has demonstrated that both the amino- and carboxy-terminal regions of this protein are necessary for the formation of filaments in the infected cell (60). Other studies have assigned an aggregation function to the amino-terminal half of p10 (63, 65), and it has been shown that this region contains a coiled-coil domain which is conserved among the different baculoviruses (66). It is tempting to speculate that p10 aggregation is the result of coiled-coil interaction, but direct evidence for this hypothesis is lacking. The precise role of the carboxy terminus of p10 is still unclear, although it has been proposed to interact with tubulin (11). Deletion of the entire p10 open reading frame (ORF) through homologous recombination produces a mutant virus which is still capable of replication both in vitro and in vivo but produces fragile polyhedra with fragmented polyhedral envelopes (26, 64, 65). The p10 protein has also been implicated in disintegration of the nuclear envelope of the host cell, and this function appears to be associated with the carboxy terminus of this protein (61, 65).Our laboratory (2) recently demonstrated that the cytoplasmic filaments, which characterize the late stages of infection by AmEPV, are composed primarily of a 156-amino-acid protein called FALPE (filament-associated late protein of EPVs). These filaments are closely associated with the spheroids and their membrane envelopes. FALPE is a phosphoprotein which migrates on sodium dodecyl sulfate (SDS)-polyacrylamide gels as a 25/27-kDa doublet. This protein also contains an unusual proline-glutamic acid repeat region spanning 20 residues in the carboxy terminus of the polypeptide. The ultrastructure and close association of this protein with the occlusion bodies of AmEPV suggested that FALPE and p10 played analogous roles during infections by the respective viruses.This article addresses the structural and functional similarities between FALPE and p10. These two viral proteins are known to be major components of filamentous structures, but it is not known whether additional viral or cellular proteins cooperate during the polymerization process. In this report, we provide insight into the mechanisms which produce filaments in cells infected with either baculoviruses or EPVs. We demonstrate that p10 and FALPE can produce filaments in the absence of other viral gene products. Using the yeast two-hybrid system and a chemical cross-linking agent, we obtained evidence for self-association of either FALPE or p10. Finally, the polypeptide regions of FALPE and p10 which are required for self-association and subsequent filament formation are mapped.