cAMP activation of PKA defines an ancient signaling mechanism

Abstract

allostery ͉ NMR ͉ cyclic nucleotide binding domain T he cAMP binding domain (CBD) and cAMP are conserved from bacteria to humans as a ubiquitous signaling mechanism to translate extracellular stress signals into appropriate biological responses (1). The major receptor for cAMP in higher eukaryotes, cAMP-dependent PKA (2), is ubiquitous in mammalian cells where it exists in two forms: the inactive tetrameric holoenzyme and the active dissociated catalytic subunit (Csubunit). In the inactive holoenzyme, two C-subunits are bound to a dimeric regulatory subunit (R-subunit) (Fig. 1a). Upon binding cAMP, the R-subunits undergo a conformational change that unleashes the active C-subunits (3, 4). The Rsubunits are composed of an N-terminal dimerization/docking domain, a flexible linker that includes an autoinhibitory segment, and two tandem CBDs (CBD-A and CBD-B; Fig. 1b) (5). The CBD-A of the isoform I␣ of the R-subunit of PKA (RI␣) contains a noncontiguous ␣-subdomain, which mediates the interactions with the C-subunit and a contiguous ␤-subdomain that forms a ␤-sandwich and contains the cAMP binding pocket (i.e., the phosphate binding cassette or PBC) ( Fig. 1 c and d) (6).Crystal structures of CBD-A of RI␣ in its cAMP-(6) and C-bound (7) states have revealed two very different conformations, highlighting the conformational plasticity of this ancient domain. Although these static crystal structures define two stable end points, questions remain about the allosteric control of the reversible shuttling between the two states. How does the signal generated by cAMP binding to the PBC (Fig. 1 c and d) propagate through a long-range allosteric network that spans both ␣-and ␤-subdomains? Previous analyses (8-16) have led to the proposal of an initial allosteric model in which the ␣-and ␤-subdomains are directly coupled to each other through a salt bridge between E200 and R241 and also possibly through a hydrophobic hinge defined by the L203, I204, and Y229 side-chain cluster (9,11,12). However, mutations (17), sequence conservation analyses (1), structurebased comparisons (1), and genetic screening (18,19) indicate that several other sites, which are not accounted for by the existing model, are also likely to play an active role in the cAMP-mediated activation of PKA. To comprehensively understand this allosteric mechanism, it is therefore necessary to elucidate at high resolution how cAMP remodels the free energy landscape of CBD-A, which serves as the central controlling unit of PKA. For this purpose, we have investigated by NMR RI␣ (residues 119-244), a construct that spans both ␣-and ␤-subdomains of CBD-A and retains high-

Ligand-induced global transitions in the catalytic domain of protein kinase A

“…1B). Signals of local stabilization due to the ligand binding radiate outward toward the entire molecule via a network of contacts (48,54,55), transforming the PKA from open a closed form. During the population shift from open to closed ensemble, the local dynamics precedes the global dynamics of the entire molecule, i.e., the kinetic hierarchy is manifested.…”

Protein Kinase A Effects of an Expressed PRKAR1A Mutation Associated with Aggressive Tumors

“…Absence of amino acids 184 to 236 of the RIa protein abolished part of cAMP-binding domain A (which extends between amino acids 143 and 260) but retained cAMP-binding domain B (amino acids 261-374) and the entire amino (NH 2 ) part (22)(23)(24) including the dimerization/ docking domain. Recognition of RIa by Ca is mediated through specific amino acid interactions that include the critical Tyr 205 (26,27) that is absent in R1aD6 (Fig. 6A) are also absent in R1aD6 and play an important role in RIa-Ca binding (27).…”

“…The deletion in R1aD6 highlighted within an available model of R1a when bound to cAMP (A) and in a complex with Ca, structures were adapted from refs. 22-24. R1aD6 lacks the most conserved region of R1a (26), one that is essential for both cAMP binding and interaction with Ca. Differences in cytoplasmic and nuclear RIa (B) and Ca (C ) after introduction of the wt-R1a and R1aD6-GFP constructs in HEK293 cells.…”

cAMP‐dependent protein kinase A and the dynamics of epithelial cell surface domains: Moving membranes to keep in shape

“…Regulatory subunits of PKA bind to free catalytic subunits (α, β, γ, or PRkX) and their primary function appears to keep the catalytic subunits in an inactive state. Two molecules of cAMP, which is produced by any of the nine known mammalian adenylyl cyclases in response to activation of G proteins coupled to different membrane receptors, bind to each regulatory subunit of PKA and causes the subsequent release and activation of the catalytic subunits (10,11) .…”