flow-cytometric-analysis-bimolecular-fluorescence-complementation

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction

Among methods to study protein-protein interaction inside cells, Bimolecular Fluorescence Complementation (BiFC) is relatively simple and sensitive. BiFC ...

University of Illinois at Chicago 

Protein phosphatase 4 (PPP4) is a ubiquitous essential protein serine/threonine phosphatase found in higher eukaryotes. Coordinate variation of the levels of the catalytic subunit (PPP4c) and the regulatory subunit (R2) suggests that PPP4c and R2 form a heterodimeric core to which other regulatory subunits bind. Two proteins that specifically co-purify with Flag-epitope-tagged R2 expressed in HEK-293 cells were identified as Gemin3 and Gemin4. These two proteins have been identified previously as components of the Survival of Motor Neurons (SMN) protein complex, which is functionally defective in the hereditary disorder spinal muscular atrophy. Immuno-sedimentation of the epitope-tagged SMN protein complex from HeLa cells expressing CFP-SMN showed that the SMN protein interacts, as previously reported, with Gemin2 (SIP1), Gemin3 and Gemin4 and in addition associates with PPP4c. The SMN complex has been implicated in the assembly and maturation of small nuclear ribonucleoproteins (snRNPs). Expression of GFP-R2-PPP4c in HeLa cells enhances the temporal localisation of newly formed snRNPs, which is consistent with an association of R2-PPP4c with the SMN protein complex.

Protein phosphatase 4 interacts with the Survival of Motor Neurons complex and enhances the temporal localisation of snRNPs.

A-kinase anchoring proteins and neuronal signaling mechanisms.

The transmission of cellular signals often proceeds through multiprotein complexes where enzymes are positioned in proximity to their upstream activators and downstream substrates. In this report we demonstrate that the A-kinase anchoring protein AKAP-Lbc assembles an activation complex for the lipid-dependent enzyme protein kinase D (PKD). Using a combination of biochemical, enzymatic, and immunofluorescence techniques, we show that the anchoring protein contributes to PKD activation in two ways: it recruits an upstream kinase PKCeta and coordinates PKA phosphorylation events that release activated protein kinase D. Thus, AKAP-Lbc synchronizes PKA and PKC activities in a manner that leads to the activation of a third kinase. This configuration illustrates the utility of kinase anchoring as a mechanism to constrain the action of broad-spectrum enzymes.

AKAP-Lbc nucleates a protein kinase D activation scaffold.

Spatiotemporal organization of cAMP signaling begins with the tight control of second messenger synthesis. In response to agonist stimulation of G protein-coupled receptors, membrane-associated adenylyl cyclases (ACs) generate cAMP that diffuses throughout the cell. The availability of cAMP activates various intracellular effectors, including protein kinase A (PKA). Specificity in PKA action is achieved by the localization of the enzyme near its substrates through association with A-kinase anchoring proteins (AKAPs). Here, we provide evidence for interactions between AKAP79/150 and ACV and ACVI. PKA anchoring facilitates the preferential phosphorylation of AC to inhibit cAMP synthesis. Real-time cellular imaging experiments show that PKA anchoring with the cAMP synthesis machinery ensures rapid termination of cAMP signaling upon activation of the kinase. This protein configuration permits the formation of a negative feedback loop that temporally regulates cAMP production.

Dynamic regulation of cAMP synthesis through anchored PKA-adenylyl cyclase V/VI complexes.

Elevated catecholamines in the heart evoke transcriptional activation of the Myocyte Enhancer Factor (MEF) pathway to induce a cellular response known as pathological myocardial hypertrophy. We have discovered that the A-Kinase Anchoring Protein (AKAP)-Lbc is upregulated in hypertrophic cardiomyocytes. It coordinates activation and movement of signaling proteins that initiate MEF2-mediated transcriptional reprogramming events. Live-cell imaging, fluorescent kinase activity reporters, and RNA interference techniques show that AKAP-Lbc couples activation of protein kinase D (PKD) with the phosphorylation-dependent nuclear export of the class II histone deacetylase HDAC5. These studies uncover a role for AKAP-Lbc in which increased expression of the anchoring protein selectively amplifies a signaling pathway that drives cardiac myocytes toward a pathophysiological outcome.

AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway.

Protein scaffold complexes are a key mechanism by which a common signaling pathway can serve many different functions. Sequestering a signaling enzyme to a specific subcellular environment not only ensures that the enzyme is near its relevant targets, but also segregates this activity to prevent indiscriminate phosphorylation of other substrates. One family of diverse, well-studied scaffolding proteins are the A-kinase anchoring proteins (AKAPs). These anchoring proteins form multi-protein complexes that integrate cAMP signaling with other pathways and signaling events. In this review, we focus on recent advances in the elucidation of AKAP function.

A-kinase anchoring proteins: from protein complexes to physiology and disease.

In response to injury or stress, the adult heart undergoes maladaptive changes, collectively defined as pathological cardiac remodeling. Here, we focus on the role of A-kinase anchoring proteins (AKAPs) in 3 main areas associated with cardiac remodeling and the progression of heart failure: excitation-contraction coupling, sarcomeric regulation, and induction of pathological hypertrophy. AKAPs are a diverse family of scaffold proteins that form multiprotein complexes, integrating cAMP signaling with protein kinases, phosphatases, and other effector proteins. Many AKAPs have been characterized in the heart, where they play a critical role in modulating cardiac function.

A-kinase anchoring proteins that regulate cardiac remodeling.

Members of the intersectin (ITSN) family of scaffold proteins consist of multiple modular domains, each with distinct ligand preferences. Although ITSNs were initially implicated in the regulation of endocytosis, subsequent studies have revealed a more complex role for these scaffold proteins in regulation of additional biochemical pathways. In this study, we performed a high throughput yeast two-hybrid screen to identify additional pathways regulated by these scaffolds. Although several known ITSN binding partners were identified, we isolated more than 100 new targets for the two mammalian ITSN proteins, ITSN1 and ITSN2. We present the characterization of several of these new targets which implicate ITSNs in the regulation of the Rab and Arf GTPase pathways as well as regulation of the disrupted in schizophrenia 1 (DISC1) interactome. In addition, we demonstrate that ITSN proteins form homomeric and heteromeric complexes with each other revealing an added level of complexity in the function of these evolutionarily conserved scaffolds.

Intersectin (ITSN) family of scaffolds function as molecular hubs in protein interaction networks.

A-Kinase Anchoring Proteins (AKAPs) are molecular scaffolding proteins mediating the assembly of multi-protein complexes containing cAMP-dependent protein kinase A (PKA), directing the kinase in discrete subcellular locations. Splice variants from the AKAP7 gene (AKAP15/18) are vital components of neuronal and cardiac phosphatase complexes, ion channels, cardiac Ca2+ handling and renal water transport.

Molecular evolution of A-kinase anchoring protein (AKAP)-7: implications in comparative PKA compartmentalization.

AKAP-Lbc is a scaffold protein that coordinates cardiac hypertrophic signaling.

Src homology 2 domain-containing phosphatase 2 (Shp2) is a component of the A-kinase-anchoring protein (AKAP)-Lbc complex and is inhibited by protein kinase A (PKA) under pathological hypertrophic conditions in the heart.

A-kinase anchoring proteins (AKAPs) are scaffolding molecules that coordinate and integrate G-protein signaling events to regulate development, physiology, and disease. One family member, AKAP13, encodes for multiple protein isoforms that contain binding sites for protein kinase A (PKA) and D (PKD) and an active Rho-guanine nucleotide exchange factor (Rho-GEF) domain. In mice, AKAP13 is required for development as null embryos die by embryonic day 10.5 with cardiovascular phenotypes. Additionally, the AKAP13 Rho-GEF and PKD-binding domains mediate cardiomyocyte hypertrophy in cell culture. However, the requirements for the Rho-GEF and PKD-binding domains during development and cardiac hypertrophy are unknown.

Graeme K. Carnegie

Department of Pharmacology

University of Illinois at Chicago

Flow Cytometric Analysis of Bimolecular Fluorescence Complementation: A High Throughput Quantitative Method to Study Protein-protein Interaction