CAP1 binds and activates adenylyl cyclase in mammalian cells

Abstract

CAP1 (Cyclase-Associated Protein 1) is highly conserved in evolution. Originally identified in yeast as a bifunctional protein involved in Ras-adenylyl cyclase and F-actin dynamics regulation, the adenylyl cyclase component seems to be lost in mammalian cells. Prompted by our recent identification of the Ras-like small GTPase Rap1 as a GTP-independent but geranylgeranyl-specific partner for CAP1, we hypothesized that CAP1-Rap1, similar to CAP-Ras-cyclase in yeast, might play a critical role in cAMP dynamics in mammalian cells. In this study, we report that CAP1 binds and activates mammalian adenylyl cyclase in vitro, modulates cAMP in live cells in a Rap1-dependent manner, and affects cAMP-dependent proliferation. Utilizing deletion and mutagenesis approaches, we mapped the interaction of CAP1-cyclase with CAP’s N-terminal domain involving critical leucine residues in the conserved RLE motifs and adenylyl cyclase’s conserved catalytic loops (e.g., C1a and/or C2a). When combined with a FRET-based cAMP sensor, CAP1 overexpression–knockdown strategies, and the use of constitutively active and negative regulators of Rap1, our studies highlight a critical role for CAP1-Rap1 in adenylyl cyclase regulation in live cells. Similarly, we show that CAP1 modulation significantly affected cAMP-mediated proliferation in an RLE motif–dependent manner. The combined study indicates that CAP1-cyclase-Rap1 represents a regulatory unit in cAMP dynamics and biology. Since Rap1 is an established downstream effector of cAMP, we advance the hypothesis that CAP1-cyclase-Rap1 represents a positive feedback loop that might be involved in cAMP microdomain establishment and localized signaling.

The actin-binding protein CAP1 represses MRTF-SRF–dependent gene expression in mouse cerebral cortex

“…Similar to CAP1-Δ471-474, CAP1-Δ1-35, CAP1-PP1mut, and CAP1-WH2mut normalized MRTF-SRF signaling in CAP1-KO neurons. CAP1-Δ1-35 specifically lacked OD together with two RLE repeats relevant for modulation of Gα S -stimulated adenylyl cyclase activity ( 28 , 49 ). Instead, CAP1-PP1mut is impaired in interaction with profilin or SH3 domain proteins such as tyrosine kinase ABL1 ( 27 , 50 – 52 ), and mutation of CAP1’s WH2 domain (CAP1-WH2mut) abolished binding ability to adenosine 5′-triphosphate (ATP)–G-actin ( 44 ).…”

Cyclase-associated protein 1 (CAP1) represses MRTF-SRF-dependent gene expression in the mouse cerebral cortex

“…Similar to CAP1-Δ471-474, CAP1-Δ1-35 as well as CAP1-PP1mut normalized MRTF localization and SRF activity in CAP1-KO neurons. CAP1-Δ1-35 specifically lacked OD together with two RLE motifs, which are necessary for both adenylyl cyclase binding and positive modulation of GαS-stimulated adenylyl cyclase activity in mammals (Zhang et al, 2021;Rust and Marcello, 2022), and CAP1-PP1mut is impaired in interaction with the ABP profilin or SH3 domain proteins such actin-binding protein 1 (ABP1) or tyrosine kinase ABL1 (Freeman et al, 1996;Bertling et al, 2007, Balcer, 2003Rust et al, 2020). We therefore concluded that CAP1 oligomerization, its stimulatory function towards adenylyl cyclase as well as its interaction with profilin, ABP1 or ABL1 were not important for neuronal MRTF-SRF signaling.…”

Soluble cyclase-mediated nuclear cAMP synthesis is sufficient for cell proliferation

“…Reactions were stopped in trichloroacetic acid (7.5% w/v). Product [ 3 H]-cAMP was separated from the substrate [ 3 H]-ATP by sequential column chromatography over Dowex and Alumina, as previously described 75 .…”