Dissecting cAMP Binding Domain A in the RIα Subunit of cAMP-dependent Protein Kinase

Previous neutron scattering studies elaborated the topographical relationship of the regulatory (R II␣ ) and catalytic (C ␣ ) subunits of the cAMP-dependent protein kinase. We present here the results of a set of computations that lead to an atomic model of the cAMP-dependent protein kinase heterodimer, ⌬ 1-91 R II␣ -C ␣ . The first step in the modeling utilized the crystal structures for the porcine C ␣ and bovine ⌬ 1-90 R I␣ or rat ⌬ 1-111 R II␤ , to homology-model structures of the species and isoforms that had been used in the neutron scattering experiments (bovine C ␣ subunit and murine ⌬ 1-91 R II␣ subunit, respectively). A docking procedure, constrained by the dimensions and positions of the ellipsoids in the neutron-derived R-C model as well as mutagenesis data, was used to develop "best fit" models for the heterodimer. Simulated annealing, molecular dynamics, and energy minimization were then used to refine the side chain packing at the heterodimer interface. For comparison, the calculations were done using the homology models derived from both the R I␣ and R II␤ crystal structures. Both resultant models had many similarities. Each predicted similar interfaces. The R I␣ -based model has 25% more hydrogen bonds than that based on R II␤ , with seven of these potential bonds in common. The distribution of hydrophobic, polar, and charged residues at the interface was similar for both models, with a distribution more characteristic of the exposed surface residues than those in the protein interior. The calculated interface area in each is relatively small (<2000 Å 2 ). The R I␣ -based model, however, has a significantly better fit with the scattering data and is therefore the one of distinctly higher probability. With its small interface area that has a high proportion of charged and polar residues, the complex appears poised for dissociation, and each subunit existing as a stable entity. This result is consistent with the known physiological events required for cAMP-dependent activation of the kinase.The cAMP-dependent protein kinase (PKA) 1 is a multifunctional kinase that serves as a prototype for understanding second messenger signaling and protein phosphorylation (1-3). In the absence of a cAMP signal, the enzyme is inactive and exists as a dimer of dimers, having two identical regulatory (R) and two identical catalytic (C) subunits. The C subunit has the conserved catalytic core structure that is common to the majority of protein kinases. The R subunit has a dimerization domain, followed by a pseudosubstrate sequence that inhibits C subunit activity and two in tandem cAMP-binding domains (in order from the N to C termini). When two cAMP molecules bind to each of the R subunits, the C subunit is activated, presumably via some sort of release of the pseudosubstrate sequence. The activation of the C subunit has been assumed to involve dissociation of the C subunits from the R 2 homodimer (4 -6). The strongest evidence for the dissociation of the C subunit is the fact that the C subunit, but not R 2 , is...

Section: Resultsmentioning

confidence: 99%

A Structural Model of the Catalytic Subunit-regulatory Subunit Dimeric Complex of the cAMP-dependent Protein Kinase

Tung

Walsh

Trewhella

2002

“…Each domain has its own function and also communicates with other regions of the molecule as the holoenzyme undergoes conformational changes induced by cAMP binding (29). At the N terminus of the PKA-R subunit is a dimerization domain that functions to maintain the R-subunits as a stable dimer and provides a docking site for a variety of A kinase anchoring proteins (AKAPs), thereby localizing PKA to specific subcellular locations (43)(44)(45)(46)(47). After a variable, intervening linker domain at the C terminus of the PKA-R subunits are two tandem cAMP binding domains (A and B).…”

Section: Discussionmentioning

confidence: 99%

Protein Kinase A Modulates Transforming Growth Factor-β Signaling through a Direct Interaction with Smad4 Protein

Yang

et al. 2013

Background: TGF␤ induces a Smad3-Smad4 complex and PKA-R interaction. Results: We define interaction domains between Smad4 and PKA-R required for TGF␤-mediated cell growth and EMT regulation. Conclusion: An interaction between Smad4 and PKA-R regulates TGF␤ signaling. Significance: A novel cross-talk mechanism between TGF␤ and PKA signaling is identified that is critical for execution of TGF␤-mediated cellular responses.

“…Two major interaction sites between the R and C subunits were defined previously (36,41,42). One is the autoinhibitory consensus site in the linker region of R that fills the active site cleft of the C subunit and competes for the substrate-binding site (41).…”

Section: Discussionmentioning

confidence: 99%

Differential Binding of cAMP-dependent Protein Kinase Regulatory Subunit Isoforms Iα and IIβ to the Catalytic Subunit

Cheng¹,

Phelps²,

Taylor³

2001

Self Cite

Limited trypsin digestion of type I cAMP-dependent protein kinase holoenzyme results in a proteolytic-resistant ⌬(1-72) regulatory subunit core, indicating that interaction between the regulatory and catalytic subunits extends beyond the autoinhibitory site in the R subunit at the NH 2 terminus. Sequence alignment of the two R subunit isoforms, RI and RII, reveals a significantly sequence diversity at this specific region. To determine whether this sequence diversity is functionally important for interaction with the catalytic subunit, specific mutations, R133A and D328A, are introduced into sites adjacent to the active site cleft in the catalytic subunit. While replacing Arg 133 with Ala decreases binding affinity for RII, interaction between the catalytic subunit and RI is not affected. In contrast, mutant C(D328A) showed a decrease in affinity for binding RI while maintaining similar affinities for RII as compared with the wild-type catalytic subunit. These results suggest that sequence immediately NH 2 -terminal to the consensus inhibition site in RI and RII interacts with different sites at the proximal region of the active site cleft in the catalytic subunit. These isoform-specific differences would dictate a significantly different domain organization in the type I and type II holoenzymes. The regulatory (R)1 subunits of cAMP-dependent protein kinase (PKA) are multifunctional proteins that control in a variety of ways the physiological functions of this ubiquitous protein kinase (1, 2). While the R subunits have long been recognized as the primary receptor for cAMP and the major physiological inhibitor for the catalytic (C) subunit in eukaryotic cells, it is now apparent that these highly modular proteins play other important roles as well (3). The R subunits of PKA have a well defined domain structure that consists of a dimerization domain at the NH 2 terminus followed by an autoinhibitor site and two-tandem cAMP-binding domains. While the portion of the R subunit COOH-terminal to the inhibition site is responsible for high affinity binding of the C subunit and cAMP, the remaining NH 2 terminus serves as an adaptor for binding to kinase anchoring proteins (3) and other adaptor proteins, such as the SH3-containing Grb2 (4). This segment or region of the R subunit is responsible for in vivo subcellular localization and targeting of PKA.There are two general classes of PKA, designated as type I and type II, due exclusively to differences in the R subunits, RI and RII (5-7). Four different regulatory subunits, RI␣ (8), RI␤ (9), RII␣ (10), and RII␤ (11) have been identified. Regulatory isoforms are differentially expressed in tissues (12-14), and their subcellular distribution also appears to be distinct (8 -11, 15-18). The existence of a family of protein kinase A anchoring proteins (AKAPs) that tether the type II regulatory subunits to specific subcellular structures has been well documented (3), and AKAPs for both RI and RII (19,20) as well as an RI-specific AKAP protein (21) have been identified recently...