John Chant

Rac and Cdc42 induce actin polymerization and G1 cell cycle progression independently of p65PAK and the JNK/SAPK MAP kinase cascade.

Rac and Cdc42 regulate a variety of responses in mammalian cells including formation of lamellipodia and filopodia, activation of the JNK MAP kinase cascade, and induction of G1 cell cycle progression. Rac is also one of the downstream targets required for Ras-induced malignant transformation. Rac and Cdc42 containing a Y40C effector site substitution no longer intact with the Ser/Thr kinase p65PAK and are unable to activate the JNK MAP kinase pathway. However, they still induce cytoskeletal changes and G1 cell cycle progression. Rac containing an F37A effector site substitution, on the other hand, no longer interacts with the Ser/Thr kinase p160ROCK and is unable to induce lamellipodia or G1 progression. We conclude that Rac and Cdc42 control MAP kinase pathways and actin cytoskeleton organization independently through distinct downstream targets.

Gaining confidence in high-throughput protein interaction networks

Although genome-scale technologies have benefited from statistical measures of data quality, extracting biologically relevant pathways from high-throughput proteomics data remains a challenge. Here we develop a quantitative method for evaluating proteomics data. We present a logistic regression approach that uses statistical and topological descriptors to predict the biological relevance of protein-protein interactions obtained from high-throughput screens for yeast. Other sources of information, including mRNA expression, genetic interactions and database annotations, are subsequently used to validate the model predictions without bias or cross-pollution. Novel topological statistics show hierarchical organization of the network of high-confidence interactions: protein complex interactions extend one to two links, and genetic interactions represent an even finer scale of organization. Knowledge of the maximum number of links that indicates a significant correlation between protein pairs (correlation distance) enables the integrated analysis of proteomics data with data from genetics and gene expression. The type of analysis presented will be essential for analyzing the growing amount of genomic and proteomics data in model organisms and humans.

GTPase cascades choreographing cellular behavior: movement, morphogenesis, and more.

Over the last decade we have learned that most, if not atl, cellular behaviors are influenced by GTPases. Recent work on Ras-related GTPases that regulate the cytoskele-ton has brought to our attention a new regulatory mechanism: multiple GTPase switches coupled directly in a cascade. In mammalian cells, a cascade of Cdc42 controlling Rac controlling Rho coordinates the actin cytoskeleton during cell movement. In yeast cells, a related cascade of BUD1 (RSR1) controlling CDC42 and possibly RHO proteins coordinates polarization of the cytoskeleton during cell division by budding. What is the benefit of GTPase cycles so tightly linked in a cascade? Combining GTPase switches in cascades can produce regulatory circuits of sufficient sophistication to choreograph complex cellular behaviors. In GTPase cascades, one GTPase controls the action of the next GTPase. Bifunctional linker molecules are now being discovered that directly link the actions of GTPases in these cascades. Evidence suggests that GTPase cascades are highly adaptable, with branches feeding in and out at different levels: each GTPase can be independently controlled by certain input signals, and each GTPase may produce an output independent of the activation of the other cascade members. With so many GTPases controlling different cellular processes, we anticipate that the GTPase cascade will prove to be a widespread mechanism of coordination and regulation. The Basic GTPase Switch GTPases have been found to control processes as diverse as growth control, apoptosis, translation, vesicular transport , cytoskeletal organization, and nuclear import (Bo-guski and McCormick, 1993). In its simplest form, the GTPase switch has two conformations: a GTP-bound form and a GDP-bound form. In some instances, such as Ras, the GTP-bound form is active, sending a signal, while the GDP form is inactive, sending no signal. In other instances , such as ADP-ribosylation factor, cycling of a GTPase switch may govern the formation or dissolution of multisubunit protein complexes (Rothman, 1994). For almost all Ras-related GTPase switches, the rate of conversion between the GDP-bound and GTP-bound confor-mations is modulated by regulators such as guanine nucletotide exchange factors (GEFs), which stimulate the replacement of GDP by GTP, and GTPase-activating proteins (GAPs), which stimulate the intrinsic GTPase activity of the GTPase. For certain GTPases, additional regulatory

Human Ste20 homologue hPAK1 links GTPases to the JNK MAP kinase pathway

BACKGROUND The Rho-related GTP-binding proteins Cdc42 and Rac1 have been shown to regulate signaling pathways involved in cytoskeletal reorganization and stress-responsive JNK (Jun N-terminal kinase) activation. However, to date, the GTPase targets that mediate these effects have not been identified. PAK defines a growing family of mammalian kinases that are related to yeast Ste20 and are activated in vitro through binding to Cdc42 and Rac1 (PAK: p21 Cdc42-/Rac-activated kinase). Clues to PAK function have come from studies of Ste20, which controls the activity of the yeast mating mitogen-activated protein (MAP) kinase cascade, in response to a heterotrimeric G protein and Cdc42. RESULTS To initiate studies of mammalian Ste20-related kinases, we identified a novel human PAK isoform, hPAK1. When expressed in yeast, hPAK1 was able to replace Ste20 in the pheromone response pathway. Chemical mutagenesis of a plasmid encoding hPAK1, followed by transformation into yeast, led to the identification of a potent constitutively active hPAK1 with a substitution of a highly conserved amino-acid residue (L107F) in the Cdc42-binding domain. Expression of the hPAK1(L107F) allele in mammalian cells led to specific activation of the Jun N-terminal kinase MAP kinase pathway, but not the mechanistically related extracellular signal-regulated MAP kinase pathway. CONCLUSIONS These results demonstrate that hPAK1 is a GTPase effector controlling a downstream MAP kinase pathway in mammalian cells, as Ste20 does in yeast. Thus, PAK and Ste20 kinases play key parts in linking extracellular signals from membrane components, such as receptor-associated G proteins and Rho-related GTPases, to nuclear responses, such as transcriptional activation.

An IQGAP-related protein controls actin-ring formation and cytokinesis in yeast

BACKGROUND Proteins of the IQGAP family have been identified as candidate effectors for the Rho family of GTPases; however, little is known about their cellular functions. The domain structures of IQGAP family members make them excellent candidates as regulators of the cytoskeleton: their sequences include an actin-binding domain homologous to that found in calponin, IQ motifs for interaction with calmodulin, and a GTPase-binding domain. RESULTS The genomic sequence of Saccharomyces cerevisiae revealed a single gene encoding an IQGAP family member (denoted IQGAP-related protein: Iqg1). Iqg1 and IQGAPs share similarity along their entire length, with an amino-terminal calponin-homology (CH) domain, IQ repeats, and a conserved carboxyl terminus. In contrast to IQGAPs, Iqg1 lacks an identifiable GAP motif, a WW domain, and IR repeats, although the functions of these domains in IQGAPs are not well defined. Deletion of the IQG1 gene resulted in lethality. Cellular defects included a deficiency in cytokinesis, altered actin organization, aberrant nuclear segregation, and cell lysis. The primary defect appeared to be a cytokinesis defect, and the other problems possibly arose as a consequence of this initial defect. Consistent with a role in cytokinesis, Iqg1 co-localizes with an actin ring encircling the mother-bud neck late in the cell cycle -a putative cytokinetic ring. IQG1 overexpression resulted in premature actin-ring formation, suggesting that Iqg1 activity temporally controls formation of this structure during the cell cycle. CONCLUSIONS Yeast IQGAP-related protein, Iqg1, is an important regulator of cellular morphogenesis, inducing actin-ring formation in association with cytokinesis.

Establishment of cell polarity in yeast.

The establishment of cell polarity is a central feature of morphogenesis in many types of cells (Schnepf 1986; Horvitz and Herskowitz 1992; Rodriguez-Boulan and Nelson 1993; Shapiro 1993; Priess 1994). Polarity establishment involves selection of an axis of polarization followed by the asymmetric organization of cytoskeletal elements, membranous organdies, components of the plasma membrane, and components of the extracellular matrix or cell wall along this axis. In budding yeasts such as Saccharomyces cerevisiae, cell polarization is vividly manifested during the vegetative cell cycle by the appearance and selective growth of the bud, which depends on the highly polarized movement of secretory vesicles carrying new cell-surface material, and perhaps of the Golgi cisternae that generate such vesicles (Preuss et al. 1992), to the bud site and into the growing bud. This movement appears to depend primarily on the actin/myosin system (Bretscher et al. 1994; Welch et al. 1994; Govindan et al. 1995), but other cytoskeletal elements such as the cytoplasmic microtubules and the septin-containing neck filaments also polarize before bud emergence (Byers 1981; Kilmartin and Adams 1984; Ford and Pringle 1991; Kim et al. 1991; Snyder et al. 1991) and appear to play roles in modulating the pattern of cellsurface growth and/or in the segregation of organdies along the mother-bud axis (Adams 1984; Adams and Pringle 1984; Jacobs et al. 1988; Palmer et al. 1992; Li et al. 1993; Muhua et al. 1994). Thus, the central questions about the establishment of polarity in budding yeast cells concern how the axes of polarization (bud sites) are chosen, how this choice is communicated to the cytoskeletal systems, and how these processes are coordinated with other events in the cell cycle. Yeast cells also polarize during another phase of the life cycle: During mating, a cell polarizes its cytoskeleton and cell-surface growth toward its partner of opposite mating type (Tkacz and MacKay 1979; Byers 1981; Ford and Pringle 1986; Hasek et al. 1987; Read et al. 1992; Chenevert 1994), apparently in response to the gradient of secreted mating pheromone (Jackson and Hartwell 1990; Segall 1993; Chenevert 1994; Dorer et al. 1995).

Cell polarity in yeast

Subcellular asymmetry, cell polarity, is fundamental to the diverse specialized functions of eukaryotic cells. In yeast, cell polarization is essential to division and mating. As a result, this highly accessible experimental system serves as a paradigm for deciphering the molecular mechanisms underlying the generation of polarity. Beyond yeast, cell polarity is essential to the partitioning of cell fate in embryonic development, the generation of axons and their guidance during neuronal development, and the intimate communication between lymphocytes within the immune system. The polarization of yeast cells shares many features with that of these more complex examples, including regulation by both intrinsic and extrinsic cues, conserved regulatory molecules such as Cdc42 GTPase, and asymmetry of the cytoskeleton as its centerpiece. This review summarizes the molecular pathways governing the generation of cell polarity in yeast.

Cyk3, a novel SH3-domain protein, affects cytokinesis in yeast

Cytokinesis requires the wholesale reorganization of the cytoskeleton and secretion to complete the division of one cell into two. In the budding yeast Saccharomyces cerevisiae, the IQGAP-related protein Iqg1 (Cyk1) promotes cytokinetic actin ring formation and is required for cytokinesis and viability [1-3]. As the actin ring is not essential for cytokinesis or viability, Iqg1 must act by another mechanism [4]. To uncover this mechanism, a screen for high-copy suppressors of the iqg1 lethal phenotype was performed. CYK3 suppressed the requirement for IQG1 in viability and cytokinesis without restoration of the actin ring, demonstrating that CYK3 promotes cytokinesis through an actomyosin-ring-independent pathway. CYK3 encodes a novel SH3-domain protein that was found in association with the actin ring and the mother-bud neck. cyk3 null cells had misshapen mother-bud necks and were deficient in cytokinesis. In the cyk3 null strain, actin rearrangements associated with cytokinesis appeared normal, suggesting that the phenotype reflects a defect in secretory targeting or septal synthesis. Deletion of either cyk3 or hof1 alone results in a mild cytokinetic phenotype [5-7], but deletion of both genes resulted in lethality and a complete cytokinetic block, suggesting overlapping function. Thus, Cyk3 appears to be important for cytokinesis and acts potentially downstream of Iqg1.

Generation of cell polarity in yeast

Yeast cells constitute an excellent system for studying cell polarity. They polarize by means of internally programmed patterns of cell division; they polarize chemotropically towards a partner during mating; and they utilize polarity to segregate cell-fate determinants during division. In the past year, considerable progress has been made towards increasing our understanding of the molecular mechanisms underlying each of these processes.

Bud10p directs axial cell polarization in budding yeast and resembles a transmembrane receptor

BACKGROUND The budding yeast Saccharomyces cerevisiae can bud in two spatially programmed patterns: axial or bipolar. In the axial budding pattern, cells polarize and divide adjacent to the previous site of cell separation, in response to a cell-division remnant, which includes Bud3p, Bud4p and septin proteins. This paper investigates the role of an additional component of the cell-division remnant, Bud10p, in axial budding. RESULTS The sequence of Bud10p predicts a protein that contains a single trans-membrane domain but lacks similarity to known proteins. Subcellular fractionations confirm that Bud10p is associated with membranes. Bud10p accumulates as a patch at the bud site prior to bud formation, and then persists at the mother-bud neck as the bud grows. Towards the end of the cell cycle, the localization of Bud10p refines to a tight double ring which splits at cytokinesis into two single rings, one in each progeny cell. Each single ring remains until a new concentration of Bud10p forms at the developing axial bud site, immediately adjacent to the old ring. Certain aspects of Bud10p localization are dependent upon BUD3, suggesting a close functional interaction between Bud10p and Bud3p. CONCLUSIONS Bud10p is the first example of a transmembrane protein that controls cell polarization during budding. Because Bud10p contains a large extracellular domain, it is possible that Bud10p functions in a manner analogous to an extracellular matrix receptor. Clusters of Bud10p at the mother-bud neck formed in response to Bud3p (and possibly to an extracellular cue, such as a component of the cell wall), might facilitate the docking of downstream components that direct polarization of the cytoskeleton.