CDC42 Signaling in S. cerevisiae
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CDC42 Signaling in S. cerevisiae

The S. cerevisiae (Saccharomyces cerevisiae) CDC42 (Cell Division Control Protein-42), a member of the Rho/Rac subfamily of Ras-Like GTPases (GTP-Binding Proteins) and Ras superfamily of low molecular-weight GTPases, is essential for the control of cell polarity during the yeast cell cycle. CDC42 occurs in both soluble and particulate pools, and neither its abundance nor its distribution varied through the cell cycle. In S. cerevisiae, CDC42 is localized to the bud site early in the cell cycle and this localization is critical for the selection of the proper site for Bud Emergence and for Polarized Cell Growth. It plays an important role in multiple Actin-dependent morphogenetic events such as Bud Emergence, Mating-Projection Formation and Pseudohyphal Growth. CDC42 transduces signals to the Actin cytoskeleton to initiate and maintain Polarized Gorwth (Ref.1). The establishment of Cell Polarity is an important component of the overall process of cellular morphogenesis, and is manifested in at least two ways during the S. cerevisiae cell cycle-(1) selection of a non-random bud site at one of the two cell poles and (2) localized growth at the bud site resulting in the appearance and selective growth of the bud. The C-termini of the S. cerevisiae CDC42 consist of the sequence Cys-Xaa-Xaa-Leu (where Xaa is any amino acid); these termini are modified by the addition of a C20 Geranylgeranyl isoprenoid to the Cys residue. The Geranylgeranyl group is thought to anchor CDC42 protein to a cellular membrane, that is CDC42 is prenylated with a C20 Geranylgeranyl isoprene group at a C-terminal Cys residue, and this Prenylation is necessary for the membrane attachment of CDC42 (Ref.2).

Three dimensional crystal structures reveal four potential functional domains of CDC42, namely the GTP Binding Domain, Hydrolysis Domain, Effector Domain and GEF (Guanine Nucleotide Exchange Factor) Interaction Domain. The GTP Binding and Hydrolysis Domains are involved in Guanine Nucleotide Binding and GTP hydrolysis. The Effector or Switch-I domain is highly conserved among CDC42 proteins but diverges among closely related but not functionally homologous Rac GTPases. The current paradigm is that GTPases bind to GEFs when in the nucleotide-free or GDP-bound state and bind to GAPs (GTPase-Activating Protein) and downstream effectors when in the GTP-bound state. A predominant binding partner for the Effector domain is the CRIB (for “CDC42/Rac Interactive Binding”) domain (also known as the PBD, GBD or PAK (p21-Activated Protein Kinase) domain)), which occurs in many CDC42 downstream effectors, including the PAK family of Protein Kinases. GEF Interaction Domains helps to transduce signals between CDC42 and its GEFs and stimulate GEF-induced GDP dissociation (Ref.3). S. cerevisiae alters its morphology in response to both exogenous and endogenous signals, leading to both Bud Emergence and enlargement during the mitotic cell cycle, mating-projection (“Shmoo”) formation through the Mating/Pheromone Response pathway in response to exogenous Mating-Factor Pheromones, Pseudohyphal formation and Filamentous Growth in response to Starvation conditions, or Spore formation during meiosis. CDC42 regulates the first three processes but not Sporulation to date. The mechanism by which CDC42 regulates the generation of, and switching between, these different morphogenetic patterns is still unclear, but CDC42 interactions with the Actin cytoskeleton play a critical role in this regulation (Ref.1).

In all organisms, CDC42 is prenylated with a C20 Geranylgeranyl isoprene group at a C-terminal Cys residue, and this prenylation is necessary for the membrane attachment of CDC42. S. cerevisiae CDC42 contains the C-terminal 183 Lys-Lys-Ser-Lys-Lys-Cys-Thr-Ile-Leu sequence, which is essential for the membrane localization of CDC42. The localization of CDC42 at different sites of Polarized Growth during the S. cerevisiae cell cycle is mirrored by the localization of the Actin cytoskeletal network. Newly synthesized CDC42 is Geranylgeranylated by the CDC43-Ram2 and interacts with the RDI1 (Rho GDP-Dissociation Inhibitor) protein within the cytosol (Ref.3). Presumably, CDC42 is in a GDP-bound state at this point, because the CDC24 (Cell Division Control Protein-24) GEF is membrane bound. The CDC42-GDP-RDI1 complex interacts with a CDC24-Bem1 (Bem1 Protein) Complex at the plasmamembrane. This interaction and subsequent Guanine Nucleotide exchange leads to a loss of CDC42-RDI1 binding. The CDC24-Bem1 complex binds to the plasmamembrane either through an interaction between CDC24 and the GTP-bound Rsr1 (Ras-Related Protein-Rsr1) GTPase, which is already at the plasmamembrane at the site of incipient Bud Emergence, or through the CDC24 PH domain. CDC24 catalyzes the dissociation of GDP from CDC42 and GDP is replaced by GTP. As a result of this biochemical exchange reaction, CDC24 dissociates from both CDC42 and Bem1, which then interacts with GDP bound Rsr1, through the action of the Bud2 (Inhibitory Regulator Protein-Bud2/Cla2) GAP and Bud5 (Bud Site Selection Protein-Bud5). Released CDC24 is free to recycle to the bud site or become available for nucleotide exchange later in the cell cycle (Ref.3 & 4).

In the next step, activated GTP-bound CDC42 interacts with GIC1 (GTPase-Interacting Component-1) and/or GIC2 (GTPase-Interacting Component-2) and/or one of the family of PAK-Like kinases (Cla4 (Serine/Threonine-Protein Kinase-Cla4), Ste20, and Skm1 (Serine/Threonine-Protein Kinase-Skm1)), and this complex binds to the Bni1 (Bni1 Protein) scaffold protein (Ref.5). This interaction brings the PAK-Like kinase in close proximity to the Gin4 (Serine/Threonine-Protein Kinase-Gin4); Myo3 (Myosin-3) and/or Myo5 (Myosin-5), which bind to Bni1 through an interaction between their SH3 domains and the FH1 (Formin) domain, leading to the phosphorylation of the Ser357 residue of Myo3 and/or Myo5. Bni1 also bind to a number of other proteins that interact with the Actin cytoskeletal network, including the Pfy1 (Profilin) and the EF1Alpha (Elongation Factor 1-Alpha), through FH2 domain; Bud6 (Bud Site Selection Protein-Bud6), through Bud6 binding domain; Cmd1 (Calmodulin); and Ca2+ (Calcium) (Ref.6). This complex, along with other Actin-binding proteins like Bee1, Sla1 (Cytoskeleton Assembly Control Protein-Sla1), Bni4 (Bni4 Protein) nucleates the localized assembly of the Septins, Chitins and Myo1 (Myosin-1) rings and the subsequent polymerization of Actin at the bud tip, leading to Bud emergence and Apical Bud Growth. After Bud emergence, GIC2 is phosphorylated and ubiquitinated in a CDC42-GTP-dependent manner, leading to its degradation through CDC34 (Cell Division Control Protein-34), CDC53 (Cell Division Control Protein-53), Grr1 (Ubiquitin Ligase Complex F-Box Protein-Grr1), SKP1 (Suppressor of Kinetochore Protein-1) and Proteasome Complex (Ref.7 & 8).

After DNA replication, the Apical Growth of the bud switches to Isotropic Growth. This Apical-Isotropic switch depends on activation of the Clb1 (G2/Mitotic-Specific Cyclin-1)/Clb2 (G2/Mitotic-Specific Cyclin-2)/CDC28 (Cell Division Control Protein-28) Kinase complex, which, along with GTP-bound CDC42 and NAP1 (Nucleosome Assembly Protein), is needed for the hyperphosphorylation of Cla4 and Gin4; this leads to bud shape changes and the distribution of cortical Actin and CDC42 to the sides of enlarging buds (Ref.6). The action of one or more CDC42-GAPs (Bem3p (GTPase-Activating Protein-Bem3), RGA1 (Rho-Type GTPase-Activating Protein-1) and RGA2 (Rho-Type GTPase-Activating Protein-2)) is necessary for the Apical-Isotropic switch, leading to the conversion of CDC42-GTP to a GDP-bound state. CDC42 is activated to a GTP-bound state by CDC24 at the mother-bud neck region. Activated GTP-bound CDC42 interacts again with one or more of the PAK-Like kinases, probably Cla4, whose kinase activity peaks at G2/M, as well as the IQGAP homolog Iqg1 (Ras GTPase-Activating-Like Protein-Iqg1)/Cyk1 (Cytokinesis Protein-1) through its GRD domain. The Iqg1/Cyk1 IQGAP protein is another scaffold protein that interacts with the Cmd1 through its IQ domains as well as with Actin through its CH (Calponin Homology) domain. The activated PAK-Like Kinases then phosphorylate the Myo1, Myo2 and Myo4, which bind to Cmd1 and the Septin proteins present at the mother-bud neck region. Phosphorylation of Myo1 and/or Septin proteins occurs upon assembly in late G1. These interactions lead to the formation of a Septin-dependent Actomyosin ring at the Mother-Bud neck region and the subsequent contraction of this ring following anaphase, leading to Cytokinesis. The localization of cortical Actin at the mother-Bud neck region, following the contraction of the Actomyosin ring and Cytokinesis, is a prelude to Chs (Chitin Synthase)-dependent Septum formation and eventual cell separation (Ref.6 & 7).

It is interesting that CDC42 has not been implicated as a negative or inhibitory factor in any cellular process; therefore, its roles seem to be positive or stimulatory in nature. For instance, genetic and biochemical studies in S. cerevisiae, in cultured mammalian cells, and to a lesser extent in S. pombe, have identified a myriad of CDC42 regulators and effectors. However, little is known about the specificity of assorted GEFs, GAPs, GDIs (Guanine Nucleotide Dissociation Inhibitors), or downstream effectors or about the targeting mechanisms for CDC42 to the plasma membrane at sites of Polarized Growth in response to different signals or at different times in the cell cycle (Ref.1).