A cytoskeletal localizing domain in the cyclase-associated protein, CAP/Srv2p, regulates access to a distant SH3-binding site.
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.
Yu
J
J
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
Wang
C
C
Palmieri
S J
SJ
Haarer
B K
BK
Field
J
J
D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing; D000262:Adenylyl Cyclases / Q000378:metabolism*; D000595:Amino Acid Sequence; D001665:Binding Sites; D019086:Bridged Bicyclo Compounds, Heterocyclic / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism; D003598:Cytoskeletal Proteins; D003599:Cytoskeleton / Q000378:metabolism*; D029721:Drosophila Proteins; D005656:Fungal Proteins; D019204:GTP-Binding Proteins / Q000378:metabolism; D005947:Glucose / Q000494:pharmacology; D008840:Microfilament Proteins; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction; D013844:Thiazoles / Q000494:pharmacology; D053778:Thiazolidines; D018631:ras Proteins / Q000235:genetics; D018909:src Homology Domains / Q000235:genetics
D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; D019086:Bridged Bicyclo Compounds, Heterocyclic; C410502:Capt protein, Drosophila; D018797:Cell Cycle Proteins; D003598:Cytoskeletal Proteins; D029721:Drosophila Proteins; D005656:Fungal Proteins; D008840:Microfilament Proteins; C073751:SRV2 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013844:Thiazoles; D053778:Thiazolidines; D000242:Cyclic AMP; D019204:GTP-Binding Proteins; C489015:RAS2 protein, S cerevisiae; D018631:ras Proteins; D000262:Adenylyl Cyclases; D005947:Glucose; C037067:latrunculin A