In its physiological state, cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA) is a tetramer that contains a regulatory (R) subunit dimer and two catalytic (C) subunits. We describe here the 2.3 angstrom structure of full-length tetrameric RIIβ2:C2 holoenzyme. This structure showing a dimer of dimers provides a mechanistic understanding of allosteric activation by cAMP. The heterodimers are anchored together by an interface created by the β4–β5 loop in the RIIβ subunit, which docks onto the carboxyl-terminal tail of the adjacent C subunit, thereby forcing the C subunit into a fully closed conformation in the absence of nucleotide. Diffusion of magnesium adenosine triphosphate (ATP) into these crystals trapped not ATP, but the reaction products, adenosine diphosphate and the phosphorylated RIIβ subunit. This complex has implications for the dissociation-reassociation cycling of PKA. The quaternary structure of the RIIβ tetramer differs appreciably from our model of the RIα tetramer, confirming the small-angle x-ray scattering prediction that the structures of each PKA tetramer are different.

Cyclic GMP-dependent protein kinase (PKG) is a key mediator of the nitric oxide/cGMP signaling pathway and plays a central role in regulating cardiovascular and neuronal functions. The N-terminal ϳ50 amino acids of the kinase are required for homodimerization and association with isoform-specific PKGanchoring proteins (GKAPs), which target the kinase to specific substrates. To understand the molecular details of PKG dimerization and gain insight into its association with GKAPs, we solved a crystal structure of the PKG I␤ dimerization/docking domain. Our structure provides molecular details of this unique leucine/isoleucine zipper, revealing specific hydrophobic and ionic interactions that mediate dimerization and demonstrating the topology of the GKAP interaction surface.As the main effector of the nitric oxide/cGMP signaling cascade, cGMP-dependent protein kinase (PKG) regulates smooth muscle tone, inhibits platelet activation, and modulates neuronal functions (1). In mammalian cells, two different genes encode a soluble type I PKG and a membrane-anchored type II PKG (1). Both enzymes form homodimers through an N-terminal leucine/isoleucine zipper domain. PKG I has two splice variants (␣ and ␤) that differ in the first ϳ100 amino acids, resulting in unique dimerization and autoinhibitory domains. The leucine/isoleucine zipper domain mediates interaction with isotype-specific G-kinase-anchoring proteins (GKAPs), 2 targeting PKG I␣ and I␤ to different subcellular compartments and intracellular substrates (2); therefore, we refer to this region as the dimerization and docking (D/D) domain. The domain organization of PKG is shown in Fig. 1. The N-terminal D/D domain is followed by an inhibitory sequence (IS), tandem cyclic nucleotide binding pockets, and the catalytic domain. The D/D domain contains a distinct primary sequence, with a repeating pattern of leucines and isoleucines every seven residues (Fig. 1). This pattern is referred to as a heptad repeat, and the positions of residues are labeled a-g.Specific binding partners for PKG I␣ include the myosin phosphatase targeting subunit (MYPT1) of myosin light chain phosphatase and the regulator of G-protein signaling-2 (RGS-2) (3, 4). Phosphorylation of MYPT1 by PKG I␣ activates its phosphatase activity, leading to dephosphorylation of myosin light chain, which desensitizes the contractile apparatus response to calcium, resulting in vasorelaxation (3). RGS-2 functions as a GTPase-activating protein for G␣ q subunits of heterotrimeric G-protein complexes, and phosphorylation of RGS-2 by PKG I␣ increases its activity toward G␣ q , uncoupling downstream signaling from G␣ qlinked receptors for vasoconstrictive agents (4). Specific binding partners for PKG I␤ include the inositol triphosphate receptor-associated PKG substrate (IRAG) and the transcriptional regulator TFII-I (5, 6). Phosphorylation of IRAG by PKG I␤ inhibits 1,4,5-inositol triphosphate receptordependent calcium release from the endoplasmic reticulum, contributing to smooth muscle relaxation (7). Phosphor...

Background:The RII␤ subunit of PKA exhibits unique isoform-specific structural features. Results: The unique structural properties of RII␤ do not require the C-terminal cAMP-binding domain. Conclusion:The RII␤ linker and N-terminal cAMP-binding domain confer unique subunit structure and organization of the type II␤ holoenzyme. Significance: The subunit structure and organization of the type II␤ holoenzyme contribute to its unique isoform-specific biochemical properties and functions.

SuperParaMagnetic Relaxometry (SPMR) is a highly sensitive detection technology that can differentiate the magnetic signature of nanoparticles (NP) bound to tumor cells from unbound nanoparticles. Nanoparticles that reach and bind to the target cells are measurable by superconducting quantum interference device (SQUID) magnetometers (MRX instrument developed in house), while unbound nanoparticles such as those freely circulating in the bloodstream are not detected and bone and normal tissue do not produce any magnetic signal. We have developed a protocol to produce high precision 25nm+1nm (<7% dispersion) Fe3O4 nanoparticle core. These core nanoparticles are coated by a polymer shell functionalized with carboxylate groups. Antibody are then conjugated on the surface providing molecular targeting capabilities and PEG is also attached to the surface to reduce opsonization. In previous studies, we have demonstrated that when conjugated with anti-HER2 antibody such as Herceptin, these nanoparticles exhibited great specificity and selectivity towards HER2+ tumor cells in vitro and in vivo. In current studies, we expanded our nanoparticles applications to other type of cancers, such as ovarian cancer. The CA125 is a tissue-specific antigen expressed in ovarian cancer. It is associated with greater than 80% of epithelial ovarian neoplasms. OC125, a murine monoclonal antibody, reacts with glycosylation-dependent antigens present exclusively in the cleaved portion of the molecule. OC125 antibody is conjugated to our nanoparticles using the same strategy developed for Herceptin nanoparticles. Each nanoparticle contains one to three OC125 antibody molecules covalently attached to the surface based on ELISA analysis. Our results have shown that OC125-NP can distinguish CA125+ and CA125- cell lines, OVCAR3 and HeyA8 respectively. Positive signal can be competed out by pre-incubation with free OC125 antibody and negative cell line produce undetectable SPMR signal, demonstrating good sensitivity, specificity and selectivity. Antigen glypican-1 (GPC1) is a proteoglycan located on cell surface composed of a membrane-associated protein core anchored to the cytoplasmic membrane. GPC1 may play a functional role in the control of cell division and growth regulation. The expression of GPC1 has been found to be elevated in many cancer cells, including ovarian. Applying the same conjugation strategy developed for Herceptin NP, humanized anti-GPC1 antibody is conjugated to the nanoparticles with minor modification. GPC1-NP generate appreciable signal using SKOV3 cell line (GPC1+ ovarian cell lines) and the signal can be competed out by the presence of excess free GPC1 antibody. Together, these results suggest that in additional to breast cancer application, our antibody functionalized nanoparticle system can be developed for other targeted cancer detection, such as ovarian cancer. Citation Format: Marie Zhang, Eric Smith-Nguyen. Targeted detection of ovarian cancer using functionalized iron oxide nanoparticles. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3590.

How the dimeric PKA regulatory (RI and RII) subunits assemble into an inactive tetrameric complex with the catalytic subunits is very isoform specific. By solving structures of holoenzyme complexes, we now appreciate the global conformational changes that take place in each regulatory subunit as they release cAMP and wrap themselves around the catalytic subunit. By using monomeric deletion mutants we can map the novel isoform‐specific interactions of the inhibition sites in the RI and RII subunits. In parallel we have used small angle X‐ray scattering (SAXS) and small angle neutron scattering (SANS) to map the global architectures of the different conformational states. To define the shape and conformational dynamics of the RIIβ subunit, we have engineered a dimeric mutant, RIIβ(1‐280), that lacks the second cAMP binding domain. In contrast to the RIIα subunit, which forms a highly asymmetric complex, and RIα, where the tetramer is Y‐shaped, RIIβ undergoes a major conformational change. While the free RIIβ subunit forms an extended dimer, the RIIβ holoenzyme folds into a compact, globular conformation. Based on SAXS data, the properties required for this change are included within the RIIβ(1‐280) deletion mutant, and most likely relate to the linker that is disordered in the free RIIβ dimer. [Supported by NIH GM34921 to SST]