Journal of Biological Chemistry · 2001 · 212 citations · 61 references
Life SciencesCell BiologyMolecular BiologyCell PolarityCytoskeletonMolecular Cell BiologyCell Cycle ControlCell Cycle SignalingCell-cycle RegulationCyclin-dependent KinasesProtein Kinase CascadesScaffold ProteinsCell Polarity RegulationCytoskeletal PolarizationKinase Scaffold ProteinsMacromolecular Complex FormationPolarity Establishment MechanismsProtein Phosphorylation In Cell Cycle RegulationScaffold Protein ComplexesYeast Morphogenesis
In budding yeast cells, the cytoskeletal polarization and depolarization events that shape the bud are triggered at specific times during the cell cycle by the cyclin-dependent kinase Cdc28p. Polarity establishment also requires the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechanism whereby Cdc28p induces Cdc42p-dependent polarization is unknown. Here we show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p. However, the role of Cdc28p is indirect, and the phosphorylation appears to be catalyzed by the p21-activated kinase family member Cla4p and also depends on Cdc42p and the scaffold protein Bem1p. Expression of GTP-Cdc42p, the product of Cdc24p-mediated GDP/GTP exchange, stimulated Cdc24p phosphorylation independent of cell cycle cues, raising the possibility that the phosphorylation is part of a feedback regulatory pathway. Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can mediate complex formation between these proteins in vitro. We suggest that Bem1p acts to concentrate polarity establishment proteins at a discrete site, facilitating polarization and promoting Cdc24p phosphorylation at specific times during the cell cycle. In budding yeast cells, the cytoskeletal polarization and depolarization events that shape the bud are triggered at specific times during the cell cycle by the cyclin-dependent kinase Cdc28p. Polarity establishment also requires the small GTPase Cdc42p and its exchange factor, Cdc24p, but the mechanism whereby Cdc28p induces Cdc42p-dependent polarization is unknown. Here we show that Cdc24p becomes phosphorylated in a cell cycle-dependent manner, triggered by Cdc28p. However, the role of Cdc28p is indirect, and the phosphorylation appears to be catalyzed by the p21-activated kinase family member Cla4p and also depends on Cdc42p and the scaffold protein Bem1p. Expression of GTP-Cdc42p, the product of Cdc24p-mediated GDP/GTP exchange, stimulated Cdc24p phosphorylation independent of cell cycle cues, raising the possibility that the phosphorylation is part of a feedback regulatory pathway. Bem1p binds directly to Cdc24p, to Cla4p, and to GTP-bound Cdc42p and can mediate complex formation between these proteins in vitro. We suggest that Bem1p acts to concentrate polarity establishment proteins at a discrete site, facilitating polarization and promoting Cdc24p phosphorylation at specific times during the cell cycle. p21-activated kinase polymerase chain reaction kilobase pair hemagglutinin phosphate-buffered saline glutathioneS-transferase 1,4-piperazinediethanesulfonic acid polyacrylamide gel electrophoresis The regulated reorganization of the actin cytoskeleton is a crucial element in the cell division cycle of proliferating cells and in the determination of cell shape and cell motility of nonproliferating cells. In recent years, it has become apparent that many signaling pathways that trigger actin reorganizations involve small GTPases of the Rho family (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar). This family includes about 20% of the known Ras-related G proteins and can be subdivided into Rho, Rac, and Cdc42 subfamilies. CDC42 was first identified as a gene required for the establishment of cell polarity in Saccharomyces cerevisiae (2Adams A.E.M. Johnson D.I. Longnecker R.M. Sloat B.F. Pringle J.R. J. Cell Biol. 1990; 111: 131-142Crossref PubMed Scopus (470) Google Scholar), and subsequent studies showed that Cdc42p is also a key regulator of cell shape in a variety of other eukaryotes (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar,3Miller P.J. Johnson D.I. Mol. Cell. Biol. 1994; 14: 1075-1083Crossref PubMed Scopus (171) Google Scholar, 4Eaton S. Auvinen P. Luo L. Jan Y.N. Simons K. J. Cell Biol. 1995; 131: 151-164Crossref PubMed Scopus (168) Google Scholar, 5Drechsel D.N. Hyman A.A. Hall A. Glotzer M. Curr. Biol. 1996; 7: 12-23Abstract Full Text Full Text PDF Scopus (214) Google Scholar). A prominent role of Cdc42p in all cases appears to be the polarization of cells toward a variety of signals (e.g.polarization of haploid yeast toward mating partners (6Nern A. Arkowitz R.A. Nature. 1998; 391: 195-198Crossref PubMed Scopus (110) Google Scholar), of T cells toward antigen-presenting cells (7Stowers L. Yelon D. Berg L.J. Chant J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5027-5031Crossref PubMed Scopus (344) Google Scholar), or of fibroblasts toward wound sites (8Nobes C.D. Hall A. J. Cell Biol. 1999; 144: 1235-1244Crossref PubMed Scopus (1204) Google Scholar)). Human and yeast CDC42 are 80% identical and functionally interchangeable, suggesting that key functions of Cdc42p have been highly conserved (9Shinjo K. Koland K.G. Hart M.J. Narasimhan V. Johnson D.I. Evans T. Cerione R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9853-9857Crossref PubMed Scopus (172) Google Scholar, 10Munemitsu S. Innis M.A. Clark R. McCormick F. Ullrich A. Polakis P. Mol. Cell. Biol. 1990; 10: 5977-5982Crossref PubMed Scopus (132) Google Scholar).S. cerevisiae cells grow by budding, a process in which the rigid cell wall is locally expanded as a result of polarized secretion. Vectorial secretion involves delivery of secretory vesicles along polarized actin cables (11Pruyne D. Bretscher A. J. Cell Sci. 2000; 113: 571-585Crossref PubMed Google Scholar), which in turn are reorganized several times during the cell cycle (12Adams A.E.M. Pringle J.R. J. Cell Biol. 1984; 98: 934-945Crossref PubMed Scopus (597) Google Scholar), relocating growth zones to form a properly shaped bud. The actin reorganizations are triggered sequentially by changes in the activity of the cyclin-dependent kinase Cdc28p (13Lew D.J. Reed S.I. J. Cell Biol. 1993; 120: 1305-1320Crossref PubMed Scopus (402) Google Scholar), which together with several cyclins acts as the master regulator of cell cycle progression in yeast (14Lew D.J. Weinert T. Pringle J.R. Pringle J.R. Broach J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 607-695Google Scholar). Bud emergence is a tightly controlled process that normally occurs at specific sites dictated by positional markers that are inherited by newborn cells (15Chant J. Pringle J.R. J. Cell Biol. 1995; 129: 751-765Crossref PubMed Scopus (240) Google Scholar, 16Zahner J.E. Harkins H.A. Pringle J.R. Mol. Cell. Biol. 1996; 16: 1857-1870Crossref PubMed Scopus (193) Google Scholar). After a period of uniform growth during G1, activation of Cdc28p by G1cyclins triggers a dramatic polarization of many proteins as well as cytoskeletal elements toward the cortical site demarcated by the positional markers. Thus, a temporal signal (Cdc28p) allows a pre-existing cellular asymmetry (the positional markers) to be translated into a more general cytoskeletal asymmetry.The isolation by Hartwell and co-workers (17Hartwell L.H. Exp. Cell Res. 1971; 69: 265-276Crossref PubMed Scopus (470) Google Scholar) and Pringle and co-workers (2,18) of temperature-sensitive mutants that could not initiate bud formation at the restrictive temperature led to the identification of Cdc42p, along with Cdc24p and Bem1p, as critical regulators of cell polarization. Like most Ras superfamily members, Cdc42p exhibits slow intrinsic rates of GTP hydrolysis and GDP/GTP exchange that are accelerated by GTPase-activating proteins and GDP/GTP exchange factors, respectively. Cdc24p is a GDP/GTP exchange factor for Cdc42p (19Zheng Y. Cerione R. Bender A. J. Biol. Chem. 1994; 269: 2369-2372Abstract Full Text PDF PubMed Google Scholar), and the similarity between the phenotypes ofcdc24 and cdc42 mutants suggests that Cdc24p is both responsible for and dedicated to the GTP loading of Cdc42p. Bem1p is a putative scaffold protein containing two SH3 domains, andbem1Δ mutants exhibit a less severe loss of cell polarity than cdc42 or cdc24 mutants, so that cells can proliferate (albeit poorly) in its absence (18Bender A. Pringle J.R. Mol. Cell. Biol. 1991; 11: 1295-1305Crossref PubMed Scopus (350) Google Scholar, 20Chenevert J. Corrado K. Bender A. Pringle J. Herskowitz I. Nature. 1992; 356: 77-79Crossref PubMed Scopus (157) Google Scholar). Three putative GTPase-activating proteins for Cdc42p have also been identified, Bem3p, Rga1p, and Rga2p (21Zheng Y. Hart M.J. Shinjo K. Evans T. Bender A. Cerione R.A. J. Biol. Chem. 1993; 268: 24629-24634Abstract Full Text PDF PubMed Google Scholar, 22Stevenson B.J. Ferguson B. De Virgilio C. Bi E. Pringle J.R. Ammerer G. Sprague Jr., G.F. Genes Dev. 1995; 9: 2949-2963Crossref PubMed Scopus (102) Google Scholar) (see Saccharomyces genome data base for Rga2p), but their role in cell polarity remains unclear.During early G1, Cdc42p is distributed throughout the plasma membrane (and possibly internal membranes), but following Cdc28p activation a significant fraction of the Cdc42p in the cell is localized to a small patch of plasma membrane where the bud will form, together with Cdc24p and Bem1p (23Ziman M. Preuss D. Mulholland J. O'Brien J.M. Botstein D. Johnson D.I. Mol. Biol. Cell. 1993; 4: 1307-1316Crossref PubMed Scopus (203) Google Scholar, 24Pringle J.R. Bi E. Harkins H.A. Zahner J.E. De, V.irgilio C. Chant J. Corrado K. Fares H. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 729-744Crossref PubMed Scopus (152) Google Scholar, 25Nern A. Arkowitz R.A. J. Cell Biol. 1999; 144: 1187-1202Crossref PubMed Scopus (150) Google Scholar). Cdc42p then appears to recruit a number of effectors to that site, promoting polarization of the actin cytoskeleton, assembly of a ring of septins, and targeting of secretion toward that site (see Refs. 24Pringle J.R. Bi E. Harkins H.A. Zahner J.E. De, V.irgilio C. Chant J. Corrado K. Fares H. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 729-744Crossref PubMed Scopus (152) Google Scholar and 26Pruyne D. Bretscher A. J. Cell Sci. 2000; 113: 365-375Crossref PubMed Google Scholar for reviews). These observations suggested that Cdc28p promotes cell polarity through modulation of Cdc42p and its regulators. To elucidate these events, we undertook an analysis of the regulation of Cdc24p through the cell cycle. We found that Cdc24p underwent a cell cycle-dependent phosphorylation that appeared to occur in complexes containing Bem1p, GTP-Cdc42p, and the p21-activated kinase (PAK)1 Cla4p. The regulated reorganization of the actin cytoskeleton is a crucial element in the cell division cycle of proliferating cells and in the determination of cell shape and cell motility of nonproliferating cells. In recent years, it has become apparent that many signaling pathways that trigger actin reorganizations involve small GTPases of the Rho family (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar). This family includes about 20% of the known Ras-related G proteins and can be subdivided into Rho, Rac, and Cdc42 subfamilies. CDC42 was first identified as a gene required for the establishment of cell polarity in Saccharomyces cerevisiae (2Adams A.E.M. Johnson D.I. Longnecker R.M. Sloat B.F. Pringle J.R. J. Cell Biol. 1990; 111: 131-142Crossref PubMed Scopus (470) Google Scholar), and subsequent studies showed that Cdc42p is also a key regulator of cell shape in a variety of other eukaryotes (1Hall A. Science. 1998; 279: 509-514Crossref PubMed Scopus (5185) Google Scholar,3Miller P.J. Johnson D.I. Mol. Cell. Biol. 1994; 14: 1075-1083Crossref PubMed Scopus (171) Google Scholar, 4Eaton S. Auvinen P. Luo L. Jan Y.N. Simons K. J. Cell Biol. 1995; 131: 151-164Crossref PubMed Scopus (168) Google Scholar, 5Drechsel D.N. Hyman A.A. Hall A. Glotzer M. Curr. Biol. 1996; 7: 12-23Abstract Full Text Full Text PDF Scopus (214) Google Scholar). A prominent role of Cdc42p in all cases appears to be the polarization of cells toward a variety of signals (e.g.polarization of haploid yeast toward mating partners (6Nern A. Arkowitz R.A. Nature. 1998; 391: 195-198Crossref PubMed Scopus (110) Google Scholar), of T cells toward antigen-presenting cells (7Stowers L. Yelon D. Berg L.J. Chant J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5027-5031Crossref PubMed Scopus (344) Google Scholar), or of fibroblasts toward wound sites (8Nobes C.D. Hall A. J. Cell Biol. 1999; 144: 1235-1244Crossref PubMed Scopus (1204) Google Scholar)). Human and yeast CDC42 are 80% identical and functionally interchangeable, suggesting that key functions of Cdc42p have been highly conserved (9Shinjo K. Koland K.G. Hart M.J. Narasimhan V. Johnson D.I. Evans T. Cerione R.A. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 9853-9857Crossref PubMed Scopus (172) Google Scholar, 10Munemitsu S. Innis M.A. Clark R. McCormick F. Ullrich A. Polakis P. Mol. Cell. Biol. 1990; 10: 5977-5982Crossref PubMed Scopus (132) Google Scholar). S. cerevisiae cells grow by budding, a process in which the rigid cell wall is locally expanded as a result of polarized secretion. Vectorial secretion involves delivery of secretory vesicles along polarized actin cables (11Pruyne D. Bretscher A. J. Cell Sci. 2000; 113: 571-585Crossref PubMed Google Scholar), which in turn are reorganized several times during the cell cycle (12Adams A.E.M. Pringle J.R. J. Cell Biol. 1984; 98: 934-945Crossref PubMed Scopus (597) Google Scholar), relocating growth zones to form a properly shaped bud. The actin reorganizations are triggered sequentially by changes in the activity of the cyclin-dependent kinase Cdc28p (13Lew D.J. Reed S.I. J. Cell Biol. 1993; 120: 1305-1320Crossref PubMed Scopus (402) Google Scholar), which together with several cyclins acts as the master regulator of cell cycle progression in yeast (14Lew D.J. Weinert T. Pringle J.R. Pringle J.R. Broach J.R. Jones E.W. The Molecular and Cellular Biology of the Yeast Saccharomyces: Cell Cycle and Cell Biology. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1997: 607-695Google Scholar). Bud emergence is a tightly controlled process that normally occurs at specific sites dictated by positional markers that are inherited by newborn cells (15Chant J. Pringle J.R. J. Cell Biol. 1995; 129: 751-765Crossref PubMed Scopus (240) Google Scholar, 16Zahner J.E. Harkins H.A. Pringle J.R. Mol. Cell. Biol. 1996; 16: 1857-1870Crossref PubMed Scopus (193) Google Scholar). After a period of uniform growth during G1, activation of Cdc28p by G1cyclins triggers a dramatic polarization of many proteins as well as cytoskeletal elements toward the cortical site demarcated by the positional markers. Thus, a temporal signal (Cdc28p) allows a pre-existing cellular asymmetry (the positional markers) to be translated into a more general cytoskeletal asymmetry. The isolation by Hartwell and co-workers (17Hartwell L.H. Exp. Cell Res. 1971; 69: 265-276Crossref PubMed Scopus (470) Google Scholar) and Pringle and co-workers (2,18) of temperature-sensitive mutants that could not initiate bud formation at the restrictive temperature led to the identification of Cdc42p, along with Cdc24p and Bem1p, as critical regulators of cell polarization. Like most Ras superfamily members, Cdc42p exhibits slow intrinsic rates of GTP hydrolysis and GDP/GTP exchange that are accelerated by GTPase-activating proteins and GDP/GTP exchange factors, respectively. Cdc24p is a GDP/GTP exchange factor for Cdc42p (19Zheng Y. Cerione R. Bender A. J. Biol. Chem. 1994; 269: 2369-2372Abstract Full Text PDF PubMed Google Scholar), and the similarity between the phenotypes ofcdc24 and cdc42 mutants suggests that Cdc24p is both responsible for and dedicated to the GTP loading of Cdc42p. Bem1p is a putative scaffold protein containing two SH3 domains, andbem1Δ mutants exhibit a less severe loss of cell polarity than cdc42 or cdc24 mutants, so that cells can proliferate (albeit poorly) in its absence (18Bender A. Pringle J.R. Mol. Cell. Biol. 1991; 11: 1295-1305Crossref PubMed Scopus (350) Google Scholar, 20Chenevert J. Corrado K. Bender A. Pringle J. Herskowitz I. Nature. 1992; 356: 77-79Crossref PubMed Scopus (157) Google Scholar). Three putative GTPase-activating proteins for Cdc42p have also been identified, Bem3p, Rga1p, and Rga2p (21Zheng Y. Hart M.J. Shinjo K. Evans T. Bender A. Cerione R.A. J. Biol. Chem. 1993; 268: 24629-24634Abstract Full Text PDF PubMed Google Scholar, 22Stevenson B.J. Ferguson B. De Virgilio C. Bi E. Pringle J.R. Ammerer G. Sprague Jr., G.F. Genes Dev. 1995; 9: 2949-2963Crossref PubMed Scopus (102) Google Scholar) (see Saccharomyces genome data base for Rga2p), but their role in cell polarity remains unclear. During early G1, Cdc42p is distributed throughout the plasma membrane (and possibly internal membranes), but following Cdc28p activation a significant fraction of the Cdc42p in the cell is localized to a small patch of plasma membrane where the bud will form, together with Cdc24p and Bem1p (23Ziman M. Preuss D. Mulholland J. O'Brien J.M. Botstein D. Johnson D.I. Mol. Biol. Cell. 1993; 4: 1307-1316Crossref PubMed Scopus (203) Google Scholar, 24Pringle J.R. Bi E. Harkins H.A. Zahner J.E. De, V.irgilio C. Chant J. Corrado K. Fares H. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 729-744Crossref PubMed Scopus (152) Google Scholar, 25Nern A. Arkowitz R.A. J. Cell Biol. 1999; 144: 1187-1202Crossref PubMed Scopus (150) Google Scholar). Cdc42p then appears to recruit a number of effectors to that site, promoting polarization of the actin cytoskeleton, assembly of a ring of septins, and targeting of secretion toward that site (see Refs. 24Pringle J.R. Bi E. Harkins H.A. Zahner J.E. De, V.irgilio C. Chant J. Corrado K. Fares H. Cold Spring Harbor Symp. Quant. Biol. 1995; 60: 729-744Crossref PubMed Scopus (152) Google Scholar and 26Pruyne D. Bretscher A. J. Cell Sci. 2000; 113: 365-375Crossref PubMed Google Scholar for reviews). These observations suggested that Cdc28p promotes cell polarity through modulation of Cdc42p and its regulators. To elucidate these events, we undertook an analysis of the regulation of Cdc24p through the cell cycle. We found that Cdc24p underwent a cell cycle-dependent phosphorylation that appeared to occur in complexes containing Bem1p, GTP-Cdc42p, and the p21-activated kinase (PAK)1 Cla4p. We thank Erfei Bi for generously providing anti-Cdc24p antibody and Alan Bender, Erfei Bi, Fred Cross, Becky Drees, Doug Johnson, and John Pringle for providing strains and/or plasmids. We also thank John Pringle and members of the Lew laboratory for stimulating interactions.
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Molecular cloning: A laboratory manual
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