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.
Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104, USA.
J
J
Yu
C
C
Wang
S J
SJ
Palmieri
B K
BK
Haarer
J
J
Field
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