, followed by location-specific diacylglycerol generation. In response to UTP, phosphorylation of GolgiCKAR was sustained the longest, driven by the persistence of DAG, whereas phosphorylation of CytoCKAR was of the shortest duration, driven by high phosphatase activity. Our data reveal that the magnitude and duration of PKC signaling is location-specific and controlled by the level of phosphatase activity and persistence of DAG at each location.Cells respond dynamically to environmental cues conveyed by complex networks of signal transduction. Phosphorylation is the archetypal language that relays information from environmental stimuli throughout the cell; thus, hundreds of kinases and phosphatases exist to regulate the phosphorylation status of intracellular substrates. A delicate balance of phosphorylation and dephosphorylation underlies cellular decisions ranging from controlling global functions, such as proliferation or apoptosis, to regulating specialized functions, such as secretion of hormones (2, 3). Disturbing this balance can lead to disease states, most notably cancer (4).The protein kinase C (PKC) 2 family of Ser/Thr kinases transduces an abundance of extracellular signals that control diverse cellular functions, including differentiation, memory, and apoptosis. There are 10 mammalian isozymes of the PKC family, and they share a conserved COOH-terminal kinase core as well as an NH 2 -terminal autoinhibitory pseudosubstrate peptide that is lodged in the active site under resting conditions. PKC isoforms are classified into three subcategories (conventional, novel, and atypical) based on differing composition of their regulatory modules, which lie between the kinase core and inhibitory pseudosubstrate peptide (5). Conventional isoforms of PKC (cPKCs; ␣, ␤I, ␤II, and ␥) contain a tandem C1 repeat followed by a C2 domain, which allow them to respond to the second messengers diacylglycerol (DAG) and Ca 2ϩ , respectively. When extracellular signals stimulate phosphoinositide hydrolysis, DAG is produced and Ca 2ϩ is released. The binding of these second messengers to the regulatory domains results in translocation of cPKCs to cellular membranes. Both second messengers must be present for high affinity membrane binding, an event that provides the energy to disengage the inhibitory pseudosubstrate peptide from the active site, allowing downstream signaling (6). Novel isoforms of PKC (nPKCs; ␦, ⑀, , and ) are similarly activated by membrane binding; however, the novel C2 domain of the nPKCs cannot bind Ca 2ϩ . For these isozymes, high affinity membrane binding is achieved exclusively by the C1 domain, which compensates by having an increased affinity for DAG (7). Consequently, this subclass is regulated by DAG production but not by Ca 2ϩ release. Atypical PKCs and are unique in that they are not regulated by either DAG or Ca 2ϩ ; their regulatory region consists of an atypical C1 domain that does not bind DAG and a PB1 (Phox and Bem 1) domain, recently recognized for its role in protein-protein interactions ...

Summary 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 up-regulated 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 towards a pathophysiological outcome.

The serine/threonine kinase protein kinase B (PKB)/ Akt is a critical regulator of insulin signaling, cell survival, and oncogenesis. The activation mechanisms of this key kinase are well characterized. In contrast, inactivation of PKB signaling by phosphatases is less well understood. To study the dynamics of PKB signaling in live cells, we generated a genetically encoded fluorescent reporter for PKB activity that reversibly responds to stimuli activating phosphatidylinositol 3-kinase. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer, allowing real-time imaging of phosphorylation catalyzed by PKB. Because of its reversibility, the reporter also allows termination of PKB signaling by phosphatases to be monitored. We found that PKB signaling in the cytosol was more rapid and more transient compared with that in the nucleus, suggesting the presence of differentially regulated phosphatase activity in these two compartments. Furthermore, targeting of the reporter to the plasma membrane, where PKB is activated, resulted in accelerated and prolonged response compared with the response in the cytosol, suggesting that release of PKB or its substrates from the membrane is required for desensitization of PKB signaling. These data reveal spatio-temporal gradients of both signal propagation and signal termination in PKB signaling. Protein kinase B (PKB)1 /Akt is a serine/threonine kinase that is the prominent mediator of pathways resulting in enhanced cell growth and cell survival. In 1991 it was identified as the transforming component of AKT8, a virus correlating with a high incidence of spontaneous lymphoma in mice, and named Akt (1). In the same year, two other groups isolated the same kinase via its homology to protein kinase A (PKA) and protein kinase C (PKC) and designated it Rac (related to the A and C kinases) (2) and PKB (3). There are three isozymes in mammals: Akt1/PKB␣, Akt2/PKB␤, and Akt3/PKB␥. While the kinase is referred to by these two names, PKB and Akt, herein it will be referred to as PKB.PKB contains an NH 2 -terminal pleckstrin homology (PH) domain followed by a kinase domain and a short COOH-terminal regulatory tail containing an activating phosphorylation site (4). PKB is activated by recruitment to membranes following stimulation of growth factor receptors. Briefly, activated growth factor receptors lead to plasma membrane recruitment of phosphatidylinositol 3-kinase (PI 3-kinase), which leads to the production of phosphatidylinositol 3,4,5-trisphosphate (PIP 3 ) at the plasma membrane. The PH domain of PKB binds to the newly formed PIP 3 resulting in translocation of the kinase to the plasma membrane (5-8). Once at the membrane, PKB becomes activated via two sequential phosphorylation steps, first by its upstream kinase PDK-1 on Thr 308 within its activation loop, and next via autophosphorylation at Ser 473 within the COOH-terminal hydrophobic motif (9 -12). PKB is maximally active when phosphorylated at both regulat...

The proto-oncogene Akt/protein kinase B (PKB) is a pivotal signal transducer for growth and survival. Growth factor stimulation leads to Akt phosphorylation at two regulatory sites (Thr-308 and Ser-473), acutely activating Akt signaling. Delineating the exact role of each regulatory site is, however, technically challenging and has remained elusive. Here, we used genetic code expansion to produce site-specifically phosphorylated Akt1 to dissect the contribution of each regulatory site to Akt1 activity. We achieved recombinant production of full-length Akt1 containing site-specific pThr and pSer residues for the first time. Our analysis of Akt1 site-specifically phosphorylated at either or both sites revealed that phosphorylation at both sites increases the apparent catalytic rate 1500-fold relative to unphosphorylated Akt1, an increase attributable primarily to phosphorylation at Thr-308. Live imaging of COS-7 cells confirmed that phosphorylation of Thr-308, but not Ser-473, is required for cellular activation of Akt. We found and in the cell that pThr-308 function cannot be mimicked with acidic residues, nor could unphosphorylated Thr-308 be mimicked by an Ala mutation. An Akt1 variant with pSer-308 achieved only partial enzymatic and cellular signaling activity, revealing a critical interaction between the γ-methyl group of pThr-308 and Cys-310 in the Akt1 active site. Thus, pThr-308 is necessary and sufficient to stimulate Akt signaling in cells, and the common use of phosphomimetics is not appropriate for studying the biology of Akt signaling. Our data also indicate that pThr-308 should be regarded as the primary diagnostic marker of Akt activity.

Cardiac m2 muscarinic acetylcholine receptors reduce heart rate by coupling to heterotrimeric (alpha beta gamma) guanine nucleotide-binding (G) proteins that activate IKACh, an inward rectifier K+ channel (IRK). Activation of the GIRK subunit of IKACh requires G beta gamma subunits; however, the structural basis of channel regulation is unknown. To determine which sequences confer G beta gamma regulation upon IRKs, we generated chimeric proteins composed of GIRK and RB-IRK2, a related, G protein-insensitive channel. Importantly, a chimeric channel containing the hydrophobic pore region of RB-IRK2 joined to the amino and carboxyl termini of GIRK exhibited voltage- and receptor-dependent activation in Xenopus oocytes. Furthermore, carboxy-terminal sequences specific to this chimera and GIRK bound G beta gamma subunits in vitro. Thus, G beta gamma may regulate IRKs by interacting with sequences adjacent to the putative channel pore.

Environmental cues are transmitted to the interior of the cell via a complex network of signaling hubs. Receptor tyrosine kinases (RTKs) and trimeric G proteins are two such major signaling hubs in eukaryotes. Conventionally, canonical signal transduction via trimeric G proteins is thought to be triggered exclusively by G protein-coupled receptors. Here we used molecular engineering to develop modular fluorescent biosensors that exploit the remarkable specificity of bimolecular recognition, i.e., of both G proteins and RTKs, and reveal the workings of a novel platform for activation of G proteins by RTKs in single living cells. Comprised of the unique modular makeup of guanidine exchange factor Gα-interacting vesicle-associated protein (GIV)/girdin, a guanidine exchange factor that links G proteins to a variety of RTKs, these biosensors provide direct evidence that RTK-GIV-Gαi ternary complexes are formed in living cells and that Gαi is transactivated within minutes after growth factor stimulation at the plasma membrane. Thus, GIV-derived biosensors provide a versatile strategy for visualizing, monitoring, and manipulating the dynamic association of Gαi with RTKs for noncanonical transactivation of G proteins in cells and illuminate a fundamental signaling event regulated by GIV during diverse cellular processes and pathophysiologic states.

Protein kinase D (PKD) regulates many diverse cellular functions in response to diacylglycerol. To monitor PKD signaling in live cells, we generated a genetically encoded fluorescent reporter for PKD activity, DKAR (D kinase activity reporter). DKAR expressed in mammalian cells undergoes reversible fluorescence resonance energy transfer changes upon activation and inhibition of endogenous PKD. Surprisingly, we find that agonist-evoked activation of PKD is driven not only by diacylglycerol production, but by Ca 2؉ . Furthermore, elevation of intracellular Ca 2؉ , in the absence of any other stimulus, is sufficient to activate PKD. Concurrent imaging of Ca 2؉ , diacylglycerol, and PKD activity reveals that thapsigargin-mediated elevation of intracellular Ca 2؉ is closely followed by a robust increase in diacylglycerol production, in turn followed by PKD activation. The Ca 2؉ -induced production of diacylglycerol and accompanying PKD activation is dependent on phospholipase C activity. These data reveal that Ca 2؉ is a major contributor to the initiation of PKD signaling through positive feedback regulation of diacylglycerol production, unveiling a new mechanism in PKD activation.Protein kinase D (PKD) 2 comprises a family of three isoforms belonging to the Ca 2ϩ /calmodulin-dependent kinase group of serine/threonine protein kinases. PKD plays a role in numerous processes, including cell proliferation, cell survival, immune cell signaling, gene expression, vesicle trafficking, and neuronal development (1). PKD transduces signals that generate the second messenger diacylglycerol (DAG). This ligand has two roles in the activation of PKD: it activates novel protein kinase C (PKC) family members, which catalyze an activating phosphorylation of PKD, and it directly binds PKD thus recruiting it to the membrane.PKD isoforms comprise a conserved catalytic core and N-terminal regulatory moiety. The regulatory region contains two cysteine-rich (C1) domains and a pleckstrin homology domain, and this region as a whole acts in an inhibitory manner on the kinase (2). C1 domains are membrane-targeting modules that typically bind DAG and the functional analogues, phorbol esters (3). They are found in a number of proteins, most notably PKC, and provide a mechanism for proteins to be reversibly recruited to membranes in response to DAG. In the case of PKD, binding to either phorbol ester or DAG results in its membrane recruitment and activation.In addition to membrane recruitment by DAG, activation of PKD requires phosphorylation at two sites within its catalytic core (4). Thus, although DAG production leads to activation of PKD, it is not simply through the C1-mediated membrane binding and removal of autoinhibition by the regulatory region by which PKD becomes active. In addition, the upstream kinases, the novel PKCs, must phosphorylate PKD within its activation loop at Ser-744 and Ser-748 to promote its activity. This phosphorylation event is the rate-limiting step in PKD activation, and once phosphorylated, PKD remains active eve...

Summary Optimal tuning of enzyme signaling is critical for cellular homeostasis. We use fluorescence resonance energy transfer reporters in live cells to follow conformational transitions that tune the affinity of a multi-domain signal transducer, protein kinase C, for optimal response to second messengers. This enzyme comprises two diacylglycerol sensors, the C1A and C1B domains, whose intrinsic affinity for ligand is sufficiently high that the enzyme would be in a ligand-engaged, active state if not for mechanisms that mask its domains. We show that both diacylglycerol sensors are exposed in newly-synthesized protein kinase C and that conformational transitions following priming phosphorylations mask the domains such that the lower affinity sensor, the C1B domain, is the primary diacylglycerol binder. Protein kinase C's conformational rearrangements serve as a paradigm for how multi-module transducers optimize their dynamic range of signaling.