IDENTIFICATION OF A CYCLASE-ASSOCIATED PROTEIN (CAP) HOMOLOG IN DICTYOSTELIUM-DISCOIDEUM AND CHARACTERIZATION OF ITS INTERACTION WITH ACTIN

In a search for novel actin binding proteins in Dictyostelium discoide um we have isolated a cDNA clone coding for a protein of approximately 50 kDa that is highly homologous to the class of adenylyl cyclase-ass ociated proteins (CAP). In Saccharomyces cerevisiae the amino-terminal part of CAP is involved in the regulation of the adenylyl cyclase whe reas the loss of the carboxyl-terminal domain results in morphological and nutritional defects. To study the interaction of Dictyostelium CA P 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 was localized to the carboxyl-terminal domain. CAP and its carboxyl-t erminal domain led to a fluorescence enhancement of pyrene-labeled G-a ctin up to 50% indicating a direct interaction, whereas the amino-term inal domain did not enhance. In polymerization as well as in viscometr ic assays the ability of the carboxyl-terminal domain to sequester act in 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), w hereas the activity of the carboxyl-terminal domain alone was not infl uenced, suggesting that the amino-terminal half of the protein is requ ired for the PIP2 modulation of the CAP function. In profilin-minus ce lls the CAP concentration is increased by approximately 73%, indicatin g that CAP may compensate some profilin functions in vivo. In migratin g D. discoideum cells CAP was enriched at anterior and posterior plasm a membrane regions. Only a weak staining of the cytoplasm was observed . In chemotactically stimulated cells the protein was very prominent i n leading fronts. The data suggest an involvement of D. discoideum CAP in microfilament reorganization near the plasma membrane in a PIP2-re gulated manner.