ABSTRACT
Rac1 is a small G-protein in the Ras superfamily that has been implicated in the control of cell growth, adhesion, and the actin-based cytoskeleton. To investigate the role of Rac1 during motile processes, we have established Dictyostelium cell lines that conditionally overexpress epitope-tagged Dictyostelium discoideum wild-type Rac1B (DdRac1B) or a mutant DdRac1B protein. Expression of endogenous levels of myc- or GFP-tagged wild-type DdRac1B had minimal effect on cellular morphologies and behaviors. By contrast, expression of a constitutively active mutant (G12-->V or Q61-->L) or a dominant negative mutant (T17-->N) generated amoebae with characteristic cellular defects. The morphological appearance of actin-containing structures, intracellular levels of F-actin, and cellular responses to chemoattractant closely paralleled the amount of active DdRac1B, indicating a role in upregulating actin cytoskeletal activities. Expression of any of the three mutants inhibited cell growth and cytokinesis, and delayed multicellular development, suggesting that DdRac1B plays important regulatory role(s) during these processes. No significant effects were observed on binding or internalization of latex beads in suspension or on intracellular membrane trafficking. Cells expressing DdRac1B-G12V exhibited defects in fluid-phase endocytosis and the longest developmental delays; DdRac1B-Q61L produced the strongest cytokinesis defect; and DdRac1B-T17N generated intermediate phenotypes. These conditionally expressed DdRac1B proteins should facilitate the identification and characterization of the Rac1 signaling pathway in an organism that is amenable to both biochemical and molecular genetic manipulations.
Subject(s)
Actins/metabolism , Cytoskeleton/physiology , Dictyostelium/physiology , Endocytosis , Neuropeptides/metabolism , rac GTP-Binding Proteins/metabolism , Actins/immunology , Animals , Cell Adhesion , Cell Division , Cell Membrane/metabolism , Cell Movement , Cells, Cultured , Chemotaxis , Dictyostelium/cytology , Dictyostelium/genetics , Dictyostelium/growth & development , Humans , Mutagenesis, Site-Directed , Neuropeptides/genetics , Recombinant Fusion Proteins , Sequence Homology , Signal Transduction , rac GTP-Binding Proteins/genetics , rac1 GTP-Binding ProteinABSTRACT
In the yeast, Saccharomyces cerevisiae, adenylyl cyclase consists of a 200-kDa catalytic subunit (CYR1) and a 70-kDa subunit (CAP/SRV2). CAP/Srv2p assists the small G protein Ras to activate adenylyl cyclase. CAP also regulates the cytoskeleton through an actin sequestering activity and is directed to cortical actin patches by a proline-rich SH3-binding site (P2). In this report we analyze the role of the actin cytoskeleton in Ras/cAMP signaling. Two alleles of CAP, L16P(Srv2) and R19T (SupC), first isolated in genetic screens for mutants that attenuate cAMP levels, reduced adenylyl cyclase binding, and cortical actin patch localization. A third mutation, L27F, also failed to localize but showed no loss of either cAMP signaling or adenylyl cyclase binding. However, all three N-terminal mutations reduced CAP-CAP multimer formation and SH3 domain binding, although the SH3-binding site is about 350 amino acids away. Finally, disruption of the actin cytoskeleton with latrunculin-A did not affect the cAMP phenotypes of the hyperactive Ras2(Val19) allele. These data identify a novel region of CAP that controls access to the SH3-binding site and demonstrate that cytoskeletal localization of CAP or an intact cytoskeleton per se is not necessary for cAMP signaling.
Subject(s)
Adenylyl Cyclases/metabolism , Cell Cycle Proteins/metabolism , Cytoskeletal Proteins , Cytoskeleton/metabolism , Drosophila Proteins , Fungal Proteins , Microfilament Proteins , Saccharomyces cerevisiae Proteins , Actins/metabolism , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Binding Sites , Bridged Bicyclo Compounds, Heterocyclic/pharmacology , Cell Cycle Proteins/genetics , Cyclic AMP/metabolism , GTP-Binding Proteins/metabolism , Glucose/pharmacology , Molecular Sequence Data , Mutation , Protein Binding , Saccharomyces cerevisiae/enzymology , Signal Transduction , Thiazoles/pharmacology , Thiazolidines , ras Proteins/genetics , src Homology Domains/geneticsABSTRACT
A significant component of polarization in budding yeast involves the regulated restructuring of the actin cytoskeleton in response to defined cellular signals. Recent evidence suggests that such cytoskeletal organization arises through the action of large protein complexes that form in response to signals from small GTP-binding proteins, such as Cdc42, Rho, and Ras. These actin-organizing complexes may be fairly diverse, but generally consist of one or more central scaffold proteins, such as those of the formin class, that bind to signaling molecules and recruit actin-binding proteins to bring about desired polarizing events.