Article |
Address correspondence to Rong Li, Dept. of Cell Biology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115. Tel.: (617) 432-0640. Fax: (617) 432-1144. E-mail: rli{at}hms.harvard.edu
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Abstract |
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Key Words: polarity; Cdc42; actin polymerization; formin; Arp2/3 complex
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Introduction |
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We have been taking a reductionist approach to the problem of cell polarity by focusing on one of the important and commonly occurring events, that is, the assembly of localized actin cytoskeletal elements. With the knowledge that activation of Cdc42p at the presumptive bud site leads to accumulation of F-actin at this site (for review see Gulli and Peter, 2001), the problem has been reduced to understanding the link between the activated Cdc42 and actin polymerization. An elegant example of such a link in metazoan organisms has been revealed by in vitro experiments using cell extracts. Activated Cdc42p and phosphatidylinositol 4,5-bisphosphate bind to Wiskott-Aldrich syndrome protein (WASP)* or neural WASP (N-WASP), exposing a COOH-terminal domain of the latter proteins, which could activate the Arp2/3 complex, leading to nucleation of new actin filaments (Rohatgi et al., 1999, 2000; Higgs and Pollard, 2000). However, it is unclear in which physiological context this signaling pathway functions, and there is no evidence suggesting that activation of WASP or N-WASP by Cdc42p is sufficient for the establishment of a polarized actin cortex in vivo.
In yeast, assembly of cortical actin structures, the actin patches, also depends on the yeast WASP orthologue Bee1p (Lechler and Li, 1997; Li, 1997) and the Arp2/3 complex (Winter et al., 1999). Additionally, the two type I myosins, Myo3p and Myo5p, have been shown to play an important role in cortical actin assembly, and intriguingly this function seems to require the myosin motor activity (Anderson et al., 1998; Evangelista et al., 2000; Geli et al., 2000; Lechler et al., 2000). These key actin assembly factors must somehow respond to the activated Cdc42p, but this connection has not been established at the molecular level, since none of the above proteins are known to interact directly with Cdc42 in contrast to WASP and N-WASP. The aim of the present study is to define a simple set of reactions downstream of Cdc42p that could result in polarized assembly of cortical F-actin in vivo with a belief that a simple central mechanism exists underneath all the molecular complexity. We provide additional evidence that a complex containing Bee1p, Vrp1p, and type I myosins has the functional premise to be an important target of Cdc42p in the induction of local actin polymerization. We show that the localization and activity of this complex are regulated by Cdc42p through concerted actions of two Cdc42 effectors.
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Results |
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To test biochemically whether the A domain of Myo3/5p is able to activate the Arp2/3 complex in a manner similar to the A domain of Bee1p, we constructed chimeric proteins containing the WH2 domain of either Vrp1p or Bee1p fused to the NH2 terminus of the A domain of Myo3p. Both chimeras were able to activate the Arp2/3 complex to an extent similar to that by the WH2-A fragment of Bee1p when tested in a pyrene-actin polymerization assay (Fig. 1 B). To further test whether the WH2 domain of Vrp1p and the A domain of Myo3/5p can work together in a protein complex to activate the Arp2/3 complex, we used an inducible dimerization system, consisting of the proteins FRB and FKBP12, which form a tight dimer in the presence of rapamycin (Chen et al., 1995; Choi et al., 1996). The WH2 domain of Vrp1p was fused to FRB and the A domain of Myo3p to FKBP12. These two chimeras can together activate the Arp2/3 complex in the presence of rapamycin but not in its absence (Fig. 1 B). The dimer did not appear to be as potent as the Bee1 WH2-A fragment, possibly due to suboptimal orientations of the WH2 and A domains relative to each other within the dimer. Nonetheless, this result suggests that the WH2 domain of Vrp1p and the A domain of Myo3/5p are able to activate the Arp2/3 complex when present in a protein complex.
Activation of Cdc42p leads to recruitment of the Bee1pVrp1p complex to the site of cell polarization
The above data suggest that the Bee1pVrp1pmyosin-I complex plays an important role in the assembly of cortical actin filaments by activating the Arp2/3 complex. During cell polarization, the localization and/or activity of this complex is likely to be controlled by the activated Cdc42p. To test whether Bee1p is recruited to the site of cell polarization upon Cdc42p activation before actin polymerization, cells expressing Bee1-GFP and bearing the cdc28-13 mutation were synchronized in G1 at 37°C and then released from the arrest in the presence of latrunculin A (Lat-A), a compound that prevents actin polymerization (Ascough et al., 1997). When these cells were analyzed for Bee1-GFP localization by fluorescence microscopy, we observed a striking polarized distribution of Bee1-GFP at the cell cortex in the absence of F-actin (Fig. 2 A). Similar results were also obtained for Vrp1p. By comparing the kinetics of Bee1-GFP polarization with the kinetics of bud emergence in control-treated (DMSO) cells, it appears that Bee1p localization slightly precedes bud emergence, consistent with the timing of actin polarization in cells (Fig. 2 B).
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Since known Cdc42 effectors are obvious candidates for this function, we tested the ability of mutant cells deficient in each of the Cdc42p effectors to establish polarized Bee1p distribution. We observed no defects in Bee1p polarization in cells lacking both Gic1p and Gic2p, two redundant and homologous Cdc42 effectors (Brown et al., 1997; Chen et al., 1997) (Fig. 5 A). To test if PAK kinases play a role in recruiting the Bee1pVrp1p complex, we used a strain expressing a temperature-dependent degron allele of Cla4p as its sole source of PAK kinase (Holly and Blumer, 1999). We first confirmed that the shutoff phenotype in this strain can be rescued by a plasmid carrying the CLA4 gene, unlike a previous observation (Weiss et al., 2000). Similar to the gic1
gic2 double mutant, CLA4 shutoff did not affect polarized Bee1p recruitment (Table I).
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To ensure that the temperature shift itself did not contribute to the failure to polarize Bee1p, we also examined the effect of bni1 mutation during the mating factor response where Bni1p is essential for the formation of the mating projection (Lee et al., 1999; unpublished data). In
-factortreated wild-type cells, Bee1-GFP is polarized to the shmoo tip, and this polarization is further enhanced after treatment with Lat-A for 15 min (Fig. 5 E), probably due to inhibition of actin patch movement away from the shmoo tip. Consistent with a requirement of Bni1p in Bee1p polarization, Bee1-GFP, although still cortically associated, is distributed in a nonpolarized pattern in the majority of the
bni1 cells (Fig. 5 E and Table I). Polarization was also disrupted in mating cells carrying the temperature-sensitive allele of BNI1 as their sole source of formins (Table I). The results above strongly suggest that the formin-like proteins are required for Bee1p recruitment to polarization sites in response to both intracellular and extracellular signals.
We also tested whether it is Bee1p or Vrp1p that is primarily responsible for their polarized recruitment. If Bee1p is recruited through its interaction with Vrp1p, then Bee1p should no longer polarize in cells bearing the vrp1C mutation, which abrogates interaction with Bee1p (Fig. 6 A). This is indeed the case, since Bee1-GFP is not polarized in the vrp1
C strain (Fig. 6 B, a, and Table I). In contrast, Vrp1-GFP polarization occurred normally in cells bearing the bee1-
WH1 mutation, which abrogates binding to Vrp1p (Fig. 6, A and B, b, and Table I), suggesting that polarized recruitment of Vrp1p is independent of Bee1p. This result indicates that an important function of Vrp1p in polarized actin assembly is to target the Bee1pVrp1p complex to the site of polarization, consistent with the actin polarization defect observed in
vrp1 mutant cells.
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Discussion |
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Role of the Bee1p, Vrp1p, and myosin-I complex in actin nucleation
Although there is strong evidence that myosin-I interacts directly with Bee1p and Vrp1p (Anderson et al., 1998; Evangelista et al., 2000; Lechler et al., 2000), myosin-I is not present in the stable Bee1pVrp1p complex after fractionation of native extracts on the gel filtration column. This is likely due to dilution, since we have found that in concentrated samples type I myosins are associated almost stoichiometrically with Bee1p (Lechler et al., 2000). Because all observable Bee1p and Vrp1p in the extracts are in a complex with each other and because the interaction with Vrp1p has been shown to be required for myosin-I localization (Anderson et al., 1998), it is reasonable to assume that in vivo at least a population of the Bee1pVrp1p complex contains type I myosin. The interaction of the myosin-Is with the core Bee1pVrp1p complex is likely to be dynamic.
This complex would contain two sets of Arp2/3 complex activators: one in Bee1p alone and the other split between Vrp1p and myosin-I. A recent report on fission yeast myosin-I showed that the tail domain alone can activate the Arp2/3 complex (Lee et al., 2000); however, this was not observed with a similar tail region of budding yeast Myo5p (unpublished data). Instead, artificial dimerization of Myo3p A domain and Vrp1p WH2 domain was able to activate the Arp2/3 complex. Genetic redundancies between myosin-I A and Bee1p A domains (Evangelista et al., 2000; Lechler et al., 2000) and between Vrp1p WH2 and Bee1p WH2 domains (unpublished data) suggest that this mechanism of Arp2/3 complex activation can occur in vivo. At present, it is not clear why two Arp2/3 activators exist in the same complex. It is possible that the two activators function together to achieve maximum levels of local Arp2/3 complex activation. Additionally, activation of the Arp2/3 complex by two-component activators may allow the Arp2/3 complex to respond to a combination of cellular signals. Although the A domain is found only in yeast type I myosins, a recent paper reported the identification of a protein, CARMIL, in Dictyostelium, which activates and links the Arp2/3 complex to type I myosins (Jung et al., 2001). Homologues of CARMIL also exit in metazoan organisms. Thus, a myosin-I motor-containing Arp2/3-activating complex may be an important actin regulator shared by all eukaryotic cells that have dynamic actin.
Mechanism by which the Bee1Vrp1myosin-I complex is recruited to the site of cell polarization
A polarized actin cytoskeleton may potentially be established in two ways: (a) by polar movement of existing actin filament and (b) by local induction of actin polymerization. The latter is more likely because of the highly dynamic and responsive nature of the cortical actin cytoskeleton. If this were true, then the proteins that control actin assembly, in particular those that control de novo filament nucleation, should be targeted to the polarized front through interaction with polarity regulators such as Cdc42p. Indeed, we have found that the Bee1pVrp1p complex is recruited to the polarization site upon induction of the activated Cdc42p. Significantly, this process can occur in the absence of F-actin (that is, in the presence of Lat-A). Upon Lat-A washout, F-actin assembles at the site where Cdc42p and Bee1p/Vrp1p accumulate. This result supports the notion that establishment of a polarized actin distribution occurs through Cdc42 recruitment of actin polymerization factors, which locally nucleate actin filaments. This result also suggests that localization of the machinery that controls cortical actin assembly does not depend upon the actin cable-based transport system.
To elucidate the mechanism of Bee1p/Vrp1p recruitment by the activated Cdc42, we systematically tested the role of known Cdc42 effectors in this process. Two of the three sets of Cdc42 effectors, the PAKs and Gic1p/Gic2p, have been eliminated by this strategy, but the formin-like proteins are found to be required for polarized Bee1p recruitment. On the complex side, Vrp1p rather than Bee1p appeared to be involved in targeting the complex. This activity is similar to the one attributed to WIP, which was shown to recruit N-WASP to intracellular vaccinia virus, leading to actin nucleation by the Arp2/3 complex (Moreau et al., 2000). In this system, the adapter protein Nck bridges the interaction of WIP with a tyrosine-phosphorylated viral surface protein. We have not yet detected a physical interaction between the Bni1p and Vrp1p complex by coimmunoprecipitation. Since there is evidence that formin-like proteins can exist in an autoinhibited form (Alberts, 2001), it is conceivable that Bni1p in the soluble extract requires an activation event in order to interact with Vrp1p, a possibility that is currently under investigation. Since mammalian formin family proteins have also been implicated in regulating actin dynamics downstream of small GTPases (Watanabe et al., 1997), it would be interesting to test whether these proteins also play a role in localizing WASP family proteins, in particular WAVE isoforms, which themselves do not contain small GTPase-binding domains.
Myosin-I phosphorylation as a second event required for polarized actin polymerization
Our results indicate that even in a simple system such as yeast, polarized actin assembly does not occur by a linear pathway connecting signaling protein to actin assembly factors. In addition to the formin-dependent recruitment, the Bee1pVrp1pmyosin-I complex must also be activated through phosphorylation by PAKs, which is required for the motor activity of these myosins (Maruta and Korn, 1977; Wu et al., 1996) (Fig. 7). This finding is consistent with the result from actin patch reconstitution experiments in permeabilized yeast cells, which suggested an essential role for the motor activity in actin polymerization at cell cortex (Lechler et al., 2000). Motor phosphorylation is likely to occur after myosin-I is recruited to the polarization site, since the kinase responsible for this phosphorylation is recruited to the same site by Cdc42p (Peter et al., 1996). Mechanistically how the motor activity participates in polarized actin polymerization is an intensely interesting question. The motor activity may somehow be required for the activation or targeting of the Arp2/3 complex. Alternatively, it may be required after the nucleation step, for example, during filament elongation. In mammalian type I myosins, the PAK phosphorylation site is replaced by an acidic amino acid, resulting in "constitutively active" motors (Bement and Mooseker, 1995). Therefore, other activation mechanisms may exist to accompany the recruitment of actin nucleation factors in order to ensure tight spatial regulation of actin polymerization.
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Materials and methods |
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Experiments on cell polarization in the presence of Lat-A
G1 cells were collected either by growing cdc28-13 cells at 37°C or by centrifuging late log phase cells through a sucrose gradient as described (Ascough et al., 1997). 200 µM Lat-A (Miranda Sanders, University of California, Santa Cruz, CA) was then added to cells at the time of arrest release. Cells were fixed for immunofluorescence at indicated times. For the Lat-A washout experiments, the cells were washed quickly once with rich medium (YPD) before incubation in Lat-Afree media.
Fluorescence microscopy
Rhodamine-phalloidin (Molecular Probes) and immunofluorescence were carried out as described (Drubin et al., 1988; Guthrie and Fink, 1991). Anti-GFP antibody (a gift from Pam Silver and Jason Kahana, The Dana-farber Cancer Institute, Boston, MA) and FITC-conjugated donkey antirabbit secondary (Jackson ImmunoResearch Laboratories, Inc.) were used. Imaging was performed on a Nikon E600 microscope with a Mercury-100W arc lamp (Chiu Technical Corporation) and a Nikon Plan Apo 100x oil immersion objective. Image acquisition was performed with MetaMorph (Universal Imaging Corp.). Yeast strains used in this work are listed in Table II.
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Arp2/3 complex purification
The Arp2/3 complex was purified by modification of a previously established method (Egile et al., 1999). Yeast cells were lysed in UB, and an extract was made by centrifugation at 42 K for 1 h. A 55% ammonium sulfate precipitation was performed, and the pellet was dissolved and dialyzed overnight into buffer A (20 mM Tris, pH 7.5, 25 mM KCl, 1 mM MgCl2, 1 mM DTT, 0.5 mM EGTA, 0.1 mM ATP). This fraction was then incubated with GST-A(Bee1p) fragment coupled to Affigel-10 (Bio-Rad Laboratories). The beads were washed with buffer B (20 mM Tris, pH 7.5, 0.2 M KCl, 1 mM MgCl2, 1 mM DTT, 0.5 mM EGTA, 0.1 mM ATP) and eluted with buffer C (20 mM Tris, pH 7.5, 25 mM KCl, 0.2 M MgCl2, 1 mM DTT, 0.5 mM EGTA, 0.1 mM ATP). The Arp2/3 complexcontaining eluate was dialyzed into UB supplemented with 0.2 mM ATP.
Pyrene actin assembly assays
Pyrene actin assembly assays were performed in UB with 0.5 mM ATP at 30°C in an SLM Aminco Bowman series 2 luminescence spectrometer. Actin (50% pyrene labeled) concentration was 1.75 µM, the Arp2/3 complex was at 0.25 µM, and the various activators at 0.5 µM. The dimerization experiments were performed by first mixing the dimerizing pair (25 µM each protein) with or without rapamycin (50 µM) (Sigma-Aldrich) at room temperature for 15 min. The mixture was diluted to a final concentration of 0.5 µM each protein and 1 µM rapamycin in the pyrene-actin assembly assays.
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Footnotes |
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Acknowledgments |
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This work was supported by the National Institutes of Health grant GM57063-01 to R. Li, GM61345-01, and a Scholar Award from the Leukemia and Lymphoma Society of America to D. Pellman.
Submitted: 23 April 2001
Revised: 27 August 2001
Accepted: 30 August 2001
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References |
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Adams, A.E., D.I. Johnson, R.M. Longnecker, B.F. Sloat, and J.R. Pringle. 1990. CDC42 and CDC43, two additional genes involved in budding and the establishment of cell polarity in the yeast Saccharomyces cerevisiae. J. Cell Biol. 111:131142.[Abstract]
Alberts, A.S. 2001. Identification of a carboxyl-terminal Diaphanous-related formin homology protein autoregulatory domain. J. Biol. Chem. 276:28242830.
Anderson, B.L., I. Boldogh, M. Evangelista, C. Boone, L.A. Greene, and L.A. Pon. 1998. The Src homology domain 3 (SH3) of a yeast type I myosin, Myo5p, binds to verprolin and is required for targeting to sites of actin polymerization. J. Cell Biol. 141:13571370.
Ascough, K.R., J. Stryker, N. Pokala, M. Sanders, and D.G. Drubin. 1997. High rates of actin filament turnover in budding yeast and roles for actin in establishment and maintenance of cell polarity using the actin inhibitor latrunculin-A. J. Cell Biol. 137:399416.
Bement, W.M., and M.S. Mooseker. 1995. TEDS rule: a molecular rationale for differential regulation of myosins by phosphorylation of the heavy chain head. Cell Motil. Cytoskeleton. 31:8792.[Medline]
Brown, J.L., M. Jaquenoud, M.-P. Gulli, J. Chant, and M. Peter. 1997. Novel Cdc42-binding proteins Gic1 and Gic2 control cell polarity in yeast. Genes Dev. 11:29722982.
Chant, J. 1999. Cell polarity in yeast. Annu. Rev. Cell Dev. Biol. 15:365391.[Medline]
Chant, J., and J.R. Pringle. 1991. Budding and cell polarity in Saccharomyces cerevisiae. Curr. Opin. Genet. Dev. 1:342350.[Medline]
Chen, G.-C., Y.-J. Kim, and C.S.M. Chan. 1997. The Cdc42 GTPase-associated proteins Gic1 and Gic2 are required for polarized cell growth in Saccharomyces cerevisiae. Genes Dev. 11:29582971.
Chen, J., X.-F. Zheng, E.J. Brown, and S.L. Schreiber. 1995. Identification of an 11-kDa FKBP12-rapamycin-binding domain within the 289-kDa FKBP12-rapamycin-associated protein and characterization of a critical serine residue. Proc. Natl. Acad. Sci. USA. 92:49474951.[Abstract]
Choi, J., J. Chen, S.L. Schreiber, and J. Clardy. 1996. Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP. Science. 273:239242.[Abstract]
Drees, B.L., B. Sundin, E. Brazeau, J.P. Caviston, G.C. Chen, W. Guo, K.G. Kozminski, M.W. Lau, J.J. Moskow, A. Tong, et al. 2001. A protein interaction map for cell polarity development. J. Cell Biol. 154:549576.
Drubin, D.G., and W.J. Nelson. 1996. Origins of cell polarity. Cell. 84:335344.[Medline]
Drubin, D.G., K.G. Miller, and D. Botstein. 1988. Yeast actin-binding proteins: evidence for a role in morphogenesis. J. Cell Biol. 107:25512561.[Abstract]
Egile, C., T.P. Loisel, V. Laurent, R. Li, D. Pantaloni, P.J. Sansonetti, and M.F. Carlier. 1999. Activation of the CDC42 effector N-WASP by the Shigella flexneri IcsA protein promotes actin nucleation by Arp2/3 complex and bacterial actin-based motility. J. Cell Biol. 146:13191332.
Evangelista, M., K. Blundell, M.S. Longtine, C.J. Chow, N. Adames, J.R. Pringle, M. Peter, and C. Boone. 1997. Bni1p, a yeast formin linking cdc42p and the actin cytoskeleton during polarized morphogenesis. Science. 276:118122.
Evangelista, M., B.K. Klebl, A.H.Y. Tong, B.A. Webb, T. Leeuw, E. Leberer, M. Whiteway, D.Y. Thomas, and C. Boone. 2000. A role for myosin-I in actin assembly through interaction with Vrp1p, Bee1p, and the Arp2/3 complex. J. Cell Biol. 148:353362.
Geli, M.I., R. Lombardi, B. Schmelzl, and H. Riezman. 2000. An intact SH3 domain is required for myosin I-induced actin polymerization. EMBO J. 19:42814291
Gulli, M.-P., and M. Peter. 2001. Temporal and spatial regulation of Rho-type guanine-nucleotide exchange factors: the yeast perspective. Genes Dev. 15:365379.
Guthrie, C., and G.R. Fink. 1991. Guide to yeast genetics and molecular biology. Methods Enzymol. 194 pp.
Higgs, H.N., and T.D. Pollard. 1999. Regulation of actin polymerization by Arp2/3 complex and WASp/Scar proteins. J. Biol. Chem. 274:3253132534.
Higgs, H.N., and T.D. Pollard. 2000. Activation by Cdc42 and PIP(2) of Wiskott-Aldrich syndrome protein (WASp) stimulates actin nucleation by Arp2/3 complex. J. Cell Biol. 150:13111320
Holly, S.P., and K.J. Blumer. 1999. PAK family kinases regulate cell and actin polarization throughout the cell cycle of Saccharomyces cerevisiae. J. Cell Biol. 145:845856.
Imamura, H., K. Tanaka, T. Hihara, M. Umikawa, T. Kamei, K. Takahashi, T. Sasaki, and Y. Takai. 1997. Bni1p and Bnr1p: downstream targets of Rho family small G-proteins which interact with profilin and regulate actin cytoskeleton in Saccharomyces cerevisae. EMBO J. 16:27452755.
Jung G., K. Remmert, X. Wu, J.M. Volosky, and J.A. Hammer, III. 2001. The Dictyostelium CARMIL protein links capping protein and the Arp2/3 complex to type I myosins through their SH3 domains. J. Cell Biol. 153:14791497.
Lechler, T., and R. Li. 1997. In vitro reconstitution of cortical actin assembly sites in budding yeast. J. Cell Biol. 138:95103.
Lechler, T., A. Shevchenko, and R. Li. 2000. Direct involvement of yeast type I myosins in Cdc42-dependent actin polymerization. J. Cell Biol. 148:363373.
Lee, L., S.K. Klee, M. Evangelista, C. Boone, and D. Pellman. 1999. Control of mitotic spindle position by the Saccharomyces cerevisiae formin Bni1p. J. Cell Biol. 144:947961.
Lee, W.L., M. Bezanilla, and T.D. Pollard. 2000. Fission yeast myosin-I, Myo1p, stimulates actin assembly by Arp2/3 complex and shares functions with WASp. J. Cell Biol. 151:789800.
Li, R. 1997. Bee1, a yeast protein with homology to Wiskott-Aldrich syndrome protein, is critical for the assembly of cortical actin cytoskeleton. J. Cell Biol. 136:649658.
Maruta, H., and E.D. Korn. 1977. Acanthamoeba cofactor protein is a heavy chain kinase required for actin activation of the Mg2+-ATpase activity of Acanthamoeba myosin I. J. Biol. Chem. 252:83298332.[Abstract]
Moreau, V., F. Frischknecht, I. Reckmann, R. Vincentelli, G. Rabut, D. Stewart, and M. Way. 2000. A complex of N-WASP and WIP integrates signalling cascades that lead to actin polymerization. Nat. Cell Biol. 2:441448.[Medline]
Naqvi, S.N., R. Zahn, D.A. Mitchell, B.J. Stevenson, and A.L. Munn. 1998. The WASp homologue Las17p functions with the WIP homologue End5p/verprolin and is essential for endocytosis in yeast. Curr. Biol. 8:959962.[Medline]
Peter, M., A.M. Neiman, H.O. Park, M. van Lohuizen, and I. Herskowitz. 1996. Functional analysis of the interaction between the small GTP binding protein Cdc42 and the Ste20 protein kinase in yeast. EMBO J. 15:70467059.[Abstract]
Pruyne, D., and A. Bretscher. 2000a. Polarization of cell growth in yeast. I. Establishment and maintenance of polarity states. J. Cell Sci. 113:365375.
Pruyne, D., and A. Bretscher. 2000b. Polarization of cell growth in yeast. II. The role of the cortical actin cytoskeleton. J. Cell Sci. 113:571585.
Ramesh, N., I.M. Anton, J.H. Hartwig, and R.S. Geha. 1997. WIP, a protein associated with Wiskott-Aldrich syndrome protein, induces actin polymerization and redistribution in lymphoid cells. Proc. Natl. Acad. Sci. USA. 94:1467114676.
Rohatgi, R., L. Ma, H. Miki, M. Lopez, T. Kirchhausen, T. Takenawa, and M.W. Kirschner. 1999. The interaction between N-WASP and the Arp2/3 complex links Cdc42-dependent signals to actin assembly. Cell. 97:221231.[Medline]
Rohatgi, R., H.-y.H. Ho, and M.W. Kirschner. 2000. Mechanism of N-WASP activation by CDC42 and phosphatidylinositol 4,5-bisphosphate. J. Cell Biol. 150:12991310.
Sorger, P.K., and H.R. Pelham. 1987. Purification and characterization of a heat-shock element binding protein from yeast. EMBO J. 6:30353041.[Abstract]
Vaduva, G., N.C. Martin, and A.K. Hopper. 1997. Actin-binding verprolin is a polarity development protein required for the morphogenesis and function of the yeast actin cytoskeleton. J. Cell Biol. 139:18211833.
Watanabe, N., P. Madaule, T. Reid, T. Ishizaki, G. Watanabe, A. Kakizuka, Y. Saito, K. Nakao, B.M. Jockusch, and S. Narumiya. 1997. p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J. 16:30443056.
Weiss, E.L., A.C. Bishop, K.M. Shokat, and D.G. Drubin. 2000. Chemical genetic analysis of the budding-yeast p21-activated kinase Cla4p. Nat. Cell Biol. 2:677685.[Medline]
Wertman, K.F., D.G. Drubin, and D. Botstein. 1992. Systematic mutational analysis of the yeast ACT1 gene. Genetics. 132:337350.
Winter, D., T. Lechler, and R. Li. 1999. Activation of the yeast Arp2/3 complex by Bee1p, a WASP family protein. Curr. Biol. 9:501504.[Medline]
Wu, C., S.F. Lee, E. Furmaniak-Kazmierczak, G.P. Cote, D.Y. Thomas, and E. Leberer. 1996. Activation of myosin-I by members of the Ste20p protein kinase family. J. Biol. Chem. 271:3178731790.
Wu, C., V. Lytvyn, D.Y. Thomas, and E. Leberer. 1997. The phosphorylation site for Ste20p-like protein kinases is essential for the function of myosin-I in yeast. J. Biol. Chem. 272:3062330626.
Zheng, Y., R. Cerione, and A. Bender. 1994. Control of the yeast bud-site assembly GTPase Cdc42. J. Biol. Chem. 269:23692372.