Isoform-specific Differences between the Type Iα and IIα Cyclic AMP-dependent Protein Kinase Anchoring Domains Revealed by Solution NMR

Abstract

Cyclic AMP dependent protein kinase (PKA) is controlled, in part, by the subcellular localization of the enzyme (1). Discovery of dual specificity anchoring proteins (D-AKAPs) indicates that not only is the type II, but also the type I, enzyme localized (2). It appears that the type I enzyme is localized in a novel, dynamic fashion as opposed to the apparent static localization of the type II enzyme. Recently, the structure of the dimerization/ docking (D/D) domain from the type II enzyme was solved (3). This work revealed an X-type four-helix bundle motif with a hydrophobic patch that modulates AKAP interactions. To understand the dynamic versus static localization of PKA, multidimensional NMR techniques were used to investigate the structural features of the type I D/D domain. Our results indicate a conserved helix-turn-helix motif in the type I and type II D/D domains. However, important differences between the two domains are evident in the extreme NH 2 terminus: this region is extended in the type II domain, whereas it is helical in the type I protein. The NH 2 -terminal residues in RII␣ contain determinants for anchoring, and the orientation and packing of this helical element in the RI␣ structure may have profound consequences in the recognition surface presented to the AKAPs.Extracellular signals are relayed from the plasma membrane to specific intracellular targets with precision and speed. Many signaling pathways do so by altering the phosphorylation state of the target proteins. Kinases and phosphatases have broad substrate specificity, and mechanisms exist to organize and effectively concentrate the correct repertoires of enzymes into distinct signaling cascades. In the case of cAMP-dependent protein kinase (PKA), 1 the importance of signal integration and coordinated assembly of signaling cascades has been established (1). The opposing actions of adenylate cyclase and phosphodiesterases generate localized gradients of cAMP (4), which will exert its greatest influence only when concentrated pools of PKA are colocalized in an inactive conformation (5, 6). To account for this, Scott and co-workers (7) have proposed the "targeting hypothesis." This hypothesis states that phosphorylation events are not only controlled by the balance of kinase and phosphatase activity, but also by their respective subcellular localization (7). It is the regulatory subunits of PKA that mediate subcellular compartmentalization via their binding to A-Kinase anchoring proteins (AKAPs) (6,8). The anchoring of the type II PKA holoenzyme has been extensively investigated, and AKAPs have been found in centrosomes, mitochondria, Golgi, microtubules, filopodia, dendrites, and the plasma membrane (1, 9 -15). More recently, the structural basis for this high affinity interaction was revealed by solution NMR (3). The X-type four helix bundle topology in RII␣-(1-44) provides a hydrophobic interaction surface for an amphipathic helical motif in the AKAPs (3, 16).Two novel AKAPs, D-AKAP1 and D-AKAP2 (2, 17), were recently isolated and we...

Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP- dependent protein kinase

“…The dimerization domains of RI␣ and RII subunits show striking similarities in secondary structure because the N-terminal segment of both subunit types includes two ␣-helices (44). The structure and function of the distal dimerization helix (residues 49-64 in RI␣) are highly conserved between RI␣ and RII.…”

Protein kinase A-anchoring (AKAP) domains in brefeldin A-inhibited guanine nucleotide-exchange protein 2 (BIG2)

“…RI␤ has been implicated in the presynaptic regulation of hippocampal LTP͞LTD and in vivo with synaptic plasticity in the visual cortex (21-23), but no AKAP for RI␤ was identified in those systems. Three positions that are identical in RI␣ (Val-22, Ile-27, and Cys-39) and RI CE (Val-27, Ile-32, and Cys-44) govern their interaction with DAKAP1 and AKAP CE (17,20), respectively. Presence of the same residues in RI␤ (Val-22, Ile-27, and Cys-39) presumably provides a structural basis for AKAP interactions.…”

“…The experiment was replicated twice. amphipathic helix and the four-helix bundle formed by RII␣ N termini in a homodimer (17).…”

Isoform Specific Differences in Binding of a Dual-Specificity A-Kinase Anchoring Protein to Type I and Type II Regulatory Subunits of PKA

“…The His-tag was cleaved from the protein and the protein further purified using an S75-Sephadex (16/60) gel filtration column (Pharmacia) in 50 mM sodium acetate, 200 mM NaCl, 2 mM EDTA pH 4.0. Bovine RIa D/D (residues 12-61) was purified as previously described (18,19). Extinction coefficients at 280 nm were based on the method of Pace et (17).…”