title: Identification of a cyclase-associated protein (CAP) homologue in Dictyostelium discoideum and characterization of its interaction with actin.
abstract: In search for novel actin binding proteins in Dictyostelium discoideum we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-associated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whereas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CAP with actin, the complete protein and its amino-terminal and carboxyl-terminal domains were expressed in Escherichia coli and used in actin binding assays. CAP sequestered actin in a Ca2+ independent way. This activity was localized to the carboxyl-terminal domain. CAP and its carboxyl-terminal domain led to a fluorescence enhancement of pyrene-labeled G-actin up to 50% indicating a direct interaction, whereas the amino-terminal domain did not enhance. In polymerization as well as in viscometric assays the ability of the carboxyl-terminal domain to sequester actin and to prevent F-actin formation was approximately two times higher than that of intact CAP. The sequestering activity of full length CAP could be inhibited by phosphatidylinositol 4,5-bisphosphate (PIP2), whereas the activity of the carboxyl-terminal domain alone was not influenced, suggesting that the amino-terminal half of the protein is required for the PIP2 modulation of the CAP function. In profilin-minus cells the CAP concentration is increased by approximately 73%, indicating that CAP may compensate some profilin functions in vivo. In migrating D. discoideum cells CAP was enriched at anterior and posterior plasma membrane regions. Only a weak staining of the cytoplasm was observed. In chemotactically stimulated cells the protein was very prominent in leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-regulated manner.
authors.affiliation: Max-Planck-Institut für Biochemie, Martinsried, Germany.
authors.forename: U
authors.initials: U
authors.lastname: Gottwald
authors.forename: R
authors.initials: R
authors.lastname: Brokamp
authors.forename: I
authors.initials: I
authors.lastname: Karakesisoglou
authors.forename: M
authors.initials: M
authors.lastname: Schleicher
authors.forename: A A
authors.initials: AA
authors.lastname: Noegel
mesh_terms: D000199:Actins / Q000378:metabolism*; D048868:Adaptor Proteins, Signal Transducing; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D018797:Cell Cycle Proteins; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D004023:Dictyostelium / Q000254:growth & development / Q000378:metabolism*; D029721:Drosophila Proteins; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism; D051304:Profilins; D011485:Protein Binding; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C489549:CAP1 protein, human; C410502:Capt protein, Drosophila; D018797:Cell Cycle Proteins; D003285:Contractile Proteins; D003598:Cytoskeletal Proteins; D018076:DNA, Complementary; D029721:Drosophila Proteins; D005656:Fungal Proteins; D008840:Microfilament Proteins; C495738:PFN1 protein, human; D019269:Phosphatidylinositol 4,5-Diphosphate; D018129:Phosphatidylinositol Phosphates; D051304:Profilins; C073751:SRV2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins