Coiled-coil Interaction of N-terminal 36 Residues of Cyclase-associated Protein with Adenylyl Cyclase Is Sufficient for Its Function in Saccharomyces cerevisiae Ras Pathway

Abstract

In the budding yeast Saccharomyces cerevisiae, association with the 70-kDa cyclase-associated protein (CAP) is required for proper response of adenylyl cyclase to Ras proteins. We show here that a small segment comprising the N-terminal 36 amino acid residues of CAP is sufficient for association with adenylyl cyclase as well as for its function in the Ras-adenylyl cyclase pathway as assayed by the ability to confer RAS2Val-19 -dependent heat shock sensitivity to yeast cells. The CAPbinding site of adenylyl cyclase was mapped to a segment of 119 amino acid residues near its C terminus. Both of these regions contained tandem repetitions of a heptad motif ␣XX␣XXX (where ␣ represents a hydrophobic amino acid and X represents any amino acid), suggesting a coiled-coil interaction. When mutants of CAP defective in associating with adenylyl cyclase were isolated by screening of a pool of randomly mutagenized CAP, they were found to carry substitution mutations in one of the key hydrophobic residues in the heptad repeats. Furthermore, mutations of the key hydrophobic residues in the heptad repeats of adenylyl cyclase also resulted in loss of association with CAP. These results indicate the coiled-coil mechanism as a basis of the CAPadenylyl cyclase interaction.

CAS-1, a C. elegans cyclase-associated protein, is required for sarcomeric actin assembly in striated muscle

“…S1C). This region of yeast Srv2/CAP is predicted to form a coiled coil (Nishida et al, 1998), but this feature may not be conserved in C. elegans CAS-1 (see Discussion). The central region of C. elegans CAS-1 has a proline-rich region (P1) and a WASP-homology 2 (WH2) domain (supplementary material Fig.…”

“…In addition to the lack of a second proline-rich region, we noticed a potential difference between CAS-1 and other CAPs in the N-termini. The N-terminal end of yeast Srv2/CAP is predicted to form a coiled coil and implicated in its association with adenylate cyclase (Nishida et al, 1998) or oligomerization (Quintero-Monzon et al, 2009). Analysis by COILS, a prediction tool for coiled coils (Lupas et al, 1991), indicates that the N-terminal region of yeast Srv2/ CAP exhibits high probability of forming a coiled coil, but an equivalent region of C. elegans CAS-1 shows very low probability of forming a coiled coil (data not shown).…”

The role of cyclase-associated protein in regulating actin filament dynamics – more than a monomer-sequestration factor

“…1A); this region of yeast Srv2/ CAP is sufficient for binding to adenylyl cyclase, an enzyme that produces cAMP (Nishida et al, 1998) and is required for selfoligomerization of Srv2/CAP (Quintero-Monzon et al, 2009). However, this N-terminal region is unstructured in solution (Mavoungou et al, 2004) and coiled-coil formation has not been structurally demonstrated thus far.…”

Glucose Signaling in Saccharomyces cerevisiae

“…Additional signal transduction components appear to contribute to glucose signaling by contacting adenylate cyclase (Cyr1); such Cyr1 protein-protein interactions identified thus far include those with (i) an N-terminal SH3 domain in the RasGEF Cdc25 (237), (ii) the farnesylated Ras2 C terminus (73,74,184,199,338,355), (iii) the C terminus of the putative cochaperonin Sgt1 (95), (iv) the N terminus of the ancillary factor Srv2 (CAP) via coiled-coil formation (267), and (v) the RasGAP Ira1, which mediates peripheral association of adenylate cyclase with the yeast plasma membrane (241).…”