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1.  Turning Off AKT: PHLPP as a Drug Target 
Precise control of the balance between protein phosphorylation, catalyzed by protein kinases, and protein dephosphorylation, catalyzed by protein phosphatases, is essential for cellular homeostasis. Deregulation of this balance leads to pathophysiological states, driving diseases such as cancer, heart disease, and diabetes, among many others. Aberrant phosphorylation of components of the pathways that control cell growth cell survival are particularly prevalent in cancer. One of the most studied tumor suppressors in these pathways is the lipid phosphatase, PTEN, which dephosphorylates the lipid second messenger phosphatidylinositol-3,4,5-trisphosphate (PIP3), thus preventing activation of the oncogenic kinase AKT. In 2005, the discovery of a family of protein phosphatases whose members directly dephosphorylate and inactivate AKT introduced a new negative regulator of the phosphatidylinositol-3-kinase (PI3K) oncogenic pathway. PH domain Leucine-rich repeat Protein Phosphatase (PHLPP) isozymes comprise a novel tumor suppressor family whose two members, PHLPP1 and PHLPP2, are deleted as frequently as PTEN in cancers such as those of the prostate. PHLPP is thus a novel therapeutic target to suppress oncogenic pathways and is a potential candidate biomarker to stratify patients for the appropriate targeted therapeutics. This review discusses the role of PHLPP in terminating AKT signaling and how pharmacological intervention would impact this pathway.
doi:10.1146/annurev-pharmtox-011112-140338
PMCID: PMC4082184  PMID: 24392697
PHLPP; AKT; PI3K; PTEN; p53
2.  Genetically encoded fluorescent reporters to visualize protein kinase C activation in live cells 
Protein kinase C (PKC) signaling drives many important cellular processes and its dysregulation results in pathophysiologies such as cancer1. Because PKC is activated acutely and allosterically, it is difficult to monitor the cellular activity of endogenous PKC by conventional methodologies2. Rather, PKC signaling is best studied in situ using biosensors such as FRET-based reporters. We have generated several FRET-based reporters for studying PKC signaling in real time in live cells3. Using these reporters, we have demonstrated phase-locked oscillations in Ca2+ release and membrane-localized endogenous PKC activity in response to histamine4, as well as distinct signatures of endogenous PKC signaling at specific organelles in response to UTP5. Here we describe methods to image cells expressing the reporters and elaborate on data analyses, control experiments, and variations for imaging the activity of expressed PKC.
doi:10.1007/978-1-61779-160-4_17
PMCID: PMC4048020  PMID: 21870234
protein kinase C (PKC); diacylglycerol (DAG); förester or fluorescence resonance energy transfer (FRET); FRET-based reporter; C kinase activity reporter (CKAR); diacylglycerol reporter (DAGR); targeted reporter; live-cell imaging; UTP
3.  Suppression of Survival Signalling Pathways by the Phosphatase PHLPP 
The FEBS journal  2012;280(2):572-583.
Summary
The recently discovered PH (pleckstrin homology) domain Leucine rich repeat Protein Phosphatase (PHLPP) family is emerging as a central component in suppressing cell survival pathways. Originally discovered in a rational search for a phosphatase that directly dephosphorylates and inactivates Akt, PHLPP is now known to potently suppress cell survival both by inhibiting proliferative pathways and by promoting apoptotic pathways. In the first instance, PHLPP directly dephosphorylates a conserved regulatory site (termed the hydrophobic motif) on Akt, protein kinase C (PKC), and S6 kinase, thereby terminating signalling by these pro-survival kinases. In the second instance, PHLPP dephosphorylates and thus activates the pro-apoptotic kinase Mst1, thereby promoting apoptosis. PHLPP is deleted in a large number of cancers and the genetic deletion of one isozyme in a PTEN (phosphatase and tensin homolog located on chromosome 1) +/− prostate cancer model results in increased tumourigenesis, underscoring the role of PHLPP as a tumour suppressor. This review summarises the targets and cellular actions of PHLPP, with emphasis on its role as a tumour suppressor in the oncogenic PI3K (phosphoinositide 3-kinase)/Akt signalling cascade.
doi:10.1111/j.1742-4658.2012.08537.x
PMCID: PMC3770140  PMID: 22340730
PHLPP; Akt; PKC; PI3K; mTOR; PTEN; phosphatase; prostate cancer; chronic lymphocytic leukaemia
4.  Protein Kinase C Pharmacology: Refining the Toolbox 
The Biochemical journal  2013;452(2):195-209.
SYNOPSIS
Protein kinase C (PKC) has been in the limelight since the discovery three decades ago that it acts as a major receptor for the tumor-promoting phorbol esters. Phorbol esters, with their potent ability to activate two of the three classes of PKC isozymes, have remained the best pharmacological tool for directly modulating PKC activity. However, with the discovery of other phorbol ester-responsive proteins, the advent of various small-molecule and peptide modulators, and the need to distinguish isozyme-specific activity, the pharmacology of PKC has become increasingly complex. Not surprisingly, many of the compounds originally touted as direct modulators of PKC have subsequently been shown to hit many other cellular targets and, in some cases, not even directly modulate PKC. The complexities and reversals in PKC pharmacology have led to widespread confusion about the current status of the pharmacological tools available to control PKC activity. Here, we aim to clarify the cacophony in the literature regarding the current state of bona fide and discredited cellular PKC modulators, including activators, small-molecule inhibitors, and peptides, and also address the use of genetically-encoded reporters and of PKC mutants to measure the effects of these drugs on the spatiotemporal dynamics of signaling by specific isozymes.
doi:10.1042/BJ20130220
PMCID: PMC4079666  PMID: 23662807
5.  Euphohelioscopin A is a PKC activator capable of inducing macrophage differentiation 
Chemistry & biology  2012;19(8):994-1000.
SUMMARY
To identify small molecules that selectively control hematopoietic stem cell (HSC) differentiation, we performed an unbiased screen using primary human CD34+ cells. We identified a plant-derived natural product, euphohelioscopin A, capable of selectively differentiating CD34+ cells down the granulocyte/monocytic lineage. Euphohelioscopin A also inhibits proliferation and induces differentiation of the myeloid leukemia cell lines THP-1 and HL-60. Mechanistic studies revealed that euphohelioscopin A is an activator of protein kinase C (PKC), and that the pro-monocytic effects of this natural product are mediated by PKC activation. In addition to shedding new insights into normal hematopoiesis, this work may ultimately facilitate the application of stem cell therapies to a host of myeloid dysfunctions.
doi:10.1016/j.chembiol.2012.06.010
PMCID: PMC4079670  PMID: 22921066
6.  Imaging Kinase Activity at Protein Scaffolds 
Kinase signaling is under tight spatiotemporal control, with signaling hubs within the cell often coordinated by protein scaffolds. Genetically encoded kinase activity reporters afford a unique tool to interrogate the rate, amplitude, and duration of kinase signaling at specific locations throughout the cell. This protocol describes how to assay kinase activity at a protein scaffold in live cells using a fluorescence resonance energy transfer (FRET)-based kinase activity sensor for protein kinase D (PKD) as an example.
doi:10.1007/978-1-62703-622-1_10
PMCID: PMC4043534  PMID: 24052385
FRET; kinase activity reporter; DKAR; protein kinase D; scaffold protein; NHERF
7.  PHLPP regulates the development, function and molecular signaling pathways of T regulatory cells1 
Tregs have a reduced capacity to activate the PI3K/Akt pathway downstream of the TCR, and the resulting low activity of Akt is necessary for their development and function. The molecular basis for the failure of Tregs to efficiently activate Akt, however, remained unknown. We show that PH-domain Leucine-rich-repeat Protein Phosphatase (PHLPP), which dephosphorylates Akt, is up-regulated in Tregs, thus suppressing Akt activation. Tregs expressed higher levels of PHLPP than conventional T cells and knock-down of PHLPP1 restored TCR-mediated activation of Akt in Tregs. Consistent with their high Akt activity, the suppressive capacity of Tregs from PHLPP1-/- mice was significantly reduced. Moreover, the development of induced Tregs was impaired in PHLPP1-/- mice. The increased level of Akt's negative regulator, PHLPP, provides a novel mechanism used by T cells to control the Akt pathway and the first evidence for a molecular mechanism underlying the functionally essential reduction of Akt activity in Tregs.
doi:10.4049/jimmunol.1002126
PMCID: PMC4015973  PMID: 21498666
8.  Spatiotemporal Dynamics of Kinase Signaling Visualized by Targeted Reporters 
The advent of genetically encoded FRET-based kinase activity reporters has ushered in a new era of signal transduction research. Such reporters allow the direct monitoring of kinase activity in live cells at specific locations, providing unprecedented information on the spatiotemporal dynamics of kinase signaling. Specifically, FRET-sensitive conformational changes in the reporters following phosphorylation serve as a direct readout of kinase activity. These genetically encoded reporters allow not only temporal resolution of kinase activity, but also spatial resolution: by fusing appropriate targeting sequences, reporters can be positioned at specific subcellular locations. Herein we present a strategy to generate and target kinase activity reporters to discrete intracellular regions to measure kinase signaling in live cells.
doi:10.1002/9780470559277.ch090106
PMCID: PMC3146362  PMID: 21804950
kinase activity reporter; imaging; FRET; localized kinase signaling
9.  A genetically encoded fluorescent reporter reveals oscillatory phosphorylation by protein kinase C 
The Journal of Cell Biology  2003;161(5):899-909.
Signals transduced by kinases depend on the extent and duration of substrate phosphorylation. We generated genetically encoded fluorescent reporters for PKC activity that reversibly respond to stimuli activating PKC. Specifically, phosphorylation of the reporter expressed in mammalian cells causes changes in fluorescence resonance energy transfer (FRET), allowing real time imaging of phosphorylation resulting from PKC activation. Targeting of the reporter to the plasma membrane, where PKC is activated, reveals oscillatory phosphorylation in HeLa cells in response to histamine. Each oscillation in substrate phosphorylation follows a calcium oscillation with a lag of ∼10 s. Novel FRET-based reporters for PKC translocation, phosphoinositide bisphosphate conversion to IP3, and diacylglycerol show that in HeLa cells the oscillatory phosphorylations correlate with Ca2+-controlled translocation of conventional PKC to the membrane without oscillations of PLC activity or diacylglycerol. However, in MDCK cells stimulated with ATP, PLC and diacylglycerol fluctuate together with Ca2+ and phosphorylation. Thus, specificity of PKC signaling depends on the local second messenger-controlled equilibrium between kinase and phosphatase activities to result in strict calcium-controlled temporal regulation of substrate phosphorylation.
doi:10.1083/jcb.200302125
PMCID: PMC2172956  PMID: 12782683
calcium; fluorescence resonance energy transfer; oscillation; phosphatase; PKC
10.  Identification of PHLPP1 as a tumor suppressor reveals the role of feedback activation in PTEN-mutant prostate cancer progression 
Cancer cell  2011;20(2):173-186.
Summary
Hyper-activation of the PI 3-Kinase/AKT pathway is a driving force of many cancers. Here we identify the AKT-inactivating phosphatase PHLPP1 as a prostate tumor suppressor. We show that Phlpp1-loss causes neoplasia and, upon partial Pten-loss, carcinoma in mouse prostate. This genetic setting initially triggers a growth suppressive response via p53 and the Phlpp2 ortholog, and reveals spontaneous Trp53 inactivation as a condition for full-blown disease. Surprisingly, the co-deletion of PTEN and PHLPP1 in patient samples is highly restricted to metastatic disease and tightly correlated to deletion of TP53 and PHLPP2. These data establish a conceptual framework for progression of PTEN-mutant prostate cancer to life-threatening disease.
doi:10.1016/j.ccr.2011.07.013
PMCID: PMC3176728  PMID: 21840483
11.  Shedding light on local kinase activation 
BMC Biology  2012;10:61.
Phosphorylation is the predominant language of cell signaling. And, as with any common language, an abundance of dialects has evolved to convey complex information. We discuss here how biosensors are being used to decode this language, affording an unprecedented glimpse into spatio-temporal patterns of protein phosphorylation events within the cell.
doi:10.1186/1741-7007-10-61
PMCID: PMC3398854  PMID: 22805055
12.  AKAP-Lbc mobilizes a cardiac hypertrophy signaling pathway 
Molecular cell  2008;32(2):169-179.
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.
doi:10.1016/j.molcel.2008.08.030
PMCID: PMC3169907  PMID: 18951085
13.  Spinal Phosphinositide 3-Kinase-Akt-mTOR Signaling Cascades in Inflammation-Induced Hyperalgesia 
Phosphinositide 3-kinase (PI3K), Akt and their downstream kinase, mammalian target of rapamycin (mTOR) are implicated in neural plasticity. The functional linkages of this signaling cascade in spinal dorsal horn and their role in inflammatory hyperalgesia have not been elucidated. In the present work we identified the following characteristics of this cascade. i) Local inflammation led to increase in rat dorsal horn phosphorylation (activation) of Akt (pAkt) and mTOR (pmTOR), as assessed by Western blotting and immunocytochemistry. ii) Increased pAkt and pmTOR were prominent in neurons in lamina I, III and IV, while pmTOR and its downstream targets (pS6, p4EBP) were also observed in glial cells. iii) Intrathecal (IT) treatment with inhibitors to PI3K or Akt attenuated formalin-induced second phase flinching behavior, as well as carrageenan-induced thermal hyperalgesia and tactile allodynia. iv) IT rapamycin (an mTORC1 inhibitor) displayed anti-hyperalgesic effect in both inflammatory pain models. Importantly, IT wortmannin at anti-hyperalgesic doses reversed the evoked increase not only in Akt but also in mTORC1 signaling (pS6/p4EBP). v) pAkt and pmTOR are expressed in neurokinin 1 receptor (NK1R)-positive neurons in Lam I-III after peripheral inflammation. Intrathecal injection of Substance P (SP) activated this cascade (increased phosphorylation) and resulted in hyperalgesia, both of which effects were blocked by IT wortmannin and rapamycin. Together, these findings reveal that afferent inputs trigged by peripheral inflammation initiate spinal activation of PI3K-Akt-mTOR signaling pathway, a component of which participates in neuronal circuits of facilitated pain processing.
doi:10.1523/JNEUROSCI.2139-10.2011
PMCID: PMC3097059  PMID: 21307248
14.  Hydrophobic Motif Phosphorylation Is Not Required for Activation Loop Phosphorylation of p70 Ribosomal Protein S6 Kinase 1 (S6K1)* 
The Journal of Biological Chemistry  2011;286(26):23552-23558.
p70 ribosomal protein S6 kinase 1 (S6K1) is regulated by multiple phosphorylation events. Three of these sites are highly conserved among AGC kinases (cAMP dependent Protein Kinase, cGMP dependent Protein Kinase, and Protein Kinase C subfamily): the activation loop in the kinase domain, and two C-terminal sites, the turn motif and the hydrophobic motif. The common dogma has been that phosphorylation of the hydrophobic motif primes S6K1 for the phosphorylation at the activation loop by phosphoinositide-dependent protein kinase 1 (PDK1). Here, we show that the turn motif is, in fact, phosphorylated first, the activation loop second, and the hydrophobic motif is third. Specifically, biochemical analyses of a construct of S6K1 lacking the C-terminal autoinhibitory domain as well as full-length S6K1, reveals that S6K1 is constitutively phosphorylated at the turn motif when expressed in insect cells and becomes phosphorylated in vitro by purified PDK1 at the activation loop. Only the species phosphorylated at the activation loop by PDK1 gets phosphorylated at the hydrophobic motif by mammalian target of rapamycin (mTOR) in vitro. These data are consistent with a previous model in which constitutive phosphorylation of the turn motif provides the key priming step in the phosphorylation of S6K1. The data provide evidence for regulation of S6K1, where hydrophobic motif phosphorylation is not required for PDK1 to phosphorylate S6K1 at the activation loop, but instead activation loop phosphorylation of S6K1 is required for mTOR to phosphorylate the hydrophobic motif of S6K1.
doi:10.1074/jbc.M111.258004
PMCID: PMC3123118  PMID: 21561857
ATP; Cyclic Nucleotides; Enzyme Structure; Protein Kinases; Protein Kinase A (PKA); Protein Purification; Protein Structure
15.  Interaction with AKAP79 modifies the cellular pharmacology of PKC 
Molecular cell  2010;37(4):541-550.
Summary
A-Kinase Anchoring Proteins (AKAPs) coordinate cell-signaling events. AKAP79 brings together different combinations of enzyme binding partners to customize the regulation of effector proteins. In neurons muscarinic agonists mobilize an AKAP79-anchored pool of PKC that phosphorylates the KCNQ2 subunit of the M channel. This inhibits potassium permeability to enhance neuronal excitability. Using a dual fluorescent imaging/patch-clamp technique, we visualized AKAP79-anchored PKC phosphorylation of the kinase activity reporter CKAR concurrently with electrophysiological changes in KCNQ2 channels to show that AKAP79 synchronizes both signaling events to optimize the attenuation of M currents. AKAP79 also protects PKC from certain ATP competitive inhibitors. Related studies suggest that context dependent protein-protein interactions alter the susceptibility of another protein kinase, PDK1, to ATP analog inhibitors. This implies that intracellular binding partners not only couple individual molecular events in a cell signaling process but can also change the pharmacological profile of certain protein kinases.
doi:10.1016/j.molcel.2010.01.014
PMCID: PMC3014287  PMID: 20188672
16.  PHLiPPing the Switch on Akt and Protein Kinase C Signaling 
The Ser/Thr specific-phosphatase PHLPP (Pleckstrin Homology (PH) domain leucine-rich repeat protein phosphatase) provides “the brakes” for Akt and protein kinase C (PKC) signaling. The two isoforms of this recently discovered family, PHLPP1 and PHLPP2, control the amplitude and duration of signaling of Akt and PKC by catalyzing the dephosphorylation of the hydrophobic phosphorylation motif, a C-terminal phosphorylation switch that controls the activity of these kinases. Aberrant regulation of either kinase accompanies many diseases, notably diabetes and cancer. By specifically dephosphorylating the hydrophobic motif, PHLPP controls the degree of agonist-evoked signaling by Akt and the cellular levels of PKC. This review focuses on the function of PHLPP1 and PHLPP2 in modulating signaling by Akt and PKC.
doi:10.1016/j.tem.2008.04.001
PMCID: PMC2963565  PMID: 18511290
17.  PHLPP-1 Negatively Regulates Akt Activity and Survival in the Heart 
Circulation research  2010;107(4):476-484.
Rationale
The recently discovered PHLPP-1 (PH domain leucine-rich repeat protein phosphatase-1) selectively dephosphorylates Akt at Ser473 and terminates Akt signaling in cancer cells. The regulatory role of PHLPP-1 in the heart has not been considered.
Objective
To test the hypothesis that blockade/inhibition of PHLPP-1 could constitute a novel way to enhance Akt signals and provide cardioprotection.
Methods and Results
PHLPP-1 is expressed in neonatal rat ventricular myocytes (NRVMs) and in adult mouse ventricular myocytes (AMVMs). PHLPP-1 knockdown by small interfering RNA significantly enhances phosphorylation of Akt (p-Akt) at Ser473, but not at Thr308, in NRVMs stimulated with leukemia inhibitory factor (LIF). The increased phosphorylation is accompanied by greater Akt catalytic activity. PHLPP-1 knockdown enhances LIF-mediated cardioprotection against doxorubicin and also protects cardiomyocytes against H2O2. Direct Akt effects at mitochondria have been implicated in cardioprotection and mitochondria/cytosol fractionation revealed a significant enrichment of PHLPP-1 at mitochondria. The ability of PHLPP-1 knockdown to potentiate LIF-mediated increases in p-Akt at mitochondria and an accompanying increase in mitochondrial hexokinase-II was demonstrated. We generated PHLPP-1 knockout (KO) mice and demonstrate that AMVMs isolated from KO mice show potentiated p-Akt at Ser473 in response to agonists. When isolated perfused hearts are subjected to ischemia/reperfusion, p-Akt in whole-heart homogenates and in the mitochondrial fraction is significantly increased. Additionally in PHLPP-1 KO hearts, the increase in p-Akt elicited by ischemia/reperfusion is potentiated and, concomitantly, infarct size is significantly reduced.
Conclusions
These results implicate PHLPP-1 as an endogenous negative regulator of Akt activity and cell survival in the heart.
doi:10.1161/CIRCRESAHA.109.215020
PMCID: PMC2957297  PMID: 20576936
Akt; PHLPP; phosphatase; heart; protection
18.  Calcium-dependent Regulation of Protein Kinase D Revealed by a Genetically Encoded Kinase Activity Reporter* 
The Journal of biological chemistry  2006;282(9):6733-6742.
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 Ca2+. Furthermore, elevation of intracellular Ca2+, in the absence of any other stimulus, is sufficient to activate PKD. Concurrent imaging of Ca2+, diacylglycerol, and PKD activity reveals that thapsigargin-mediated elevation of intracellular Ca2+ is closely followed by a robust increase in diacylglycerol production, in turn followed by PKD activation. The Ca2+-induced production of diacylglycerol and accompanying PKD activation is dependent on phospholipase C activity. These data reveal that Ca2+ is a major contributor to the initiation of PKD signaling through positive feedback regulation of diacylglycerol production, unveiling a new mechanism in PKD activation.
doi:10.1074/jbc.M608086200
PMCID: PMC2921767  PMID: 17189263
19.  Spatio-temporal Dynamics of Protein Kinase B/Akt Signaling Revealed by a Genetically Encoded Fluorescent Reporter*◆ 
The Journal of biological chemistry  2004;280(7):5581-5587.
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. Further more, 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.
doi:10.1074/jbc.M411534200
PMCID: PMC2913970  PMID: 15583002
20.  Increased Membrane Affinity of the C1 Domain of Protein Kinase Cδ Compensates for the Lack of Involvement of Its C2 Domain in Membrane Recruitment* 
The Journal of biological chemistry  2005;281(3):1660-1669.
Protein kinase C (PKC) family members are allosterically activated following membrane recruitment by specific membrane-targeting modules. Conventional PKC isozymes are recruited to membranes by two such modules: a C1 domain, which binds diacylglycerol (DAG), and a C2 domain, which is a Ca2+-triggered phospholipid-binding module. In contrast, novel PKC isozymes respond only to DAG, despite the presence of a C2 domain. Here, we address the molecular mechanism of membrane recruitment of the novel isozyme PKCδ. We show that PKCδ and a conventional isozyme, PKCβII, bind membranes with comparable affinities. However, dissection of the contribution of individual domains to this binding revealed that, although the C2 domain is a major determinant in driving the interaction of PKCβII with membranes, the C2 domain of PKCδ does not bind membranes. Instead, the C1B domain is the determinant that drives the interaction of PKCδ with membranes. The C2 domain also does not play any detectable role in the activity or subcellular location of PKCδ in cells; in vivo imaging studies revealed that deletion of the C2 domain does not affect the stimulus-dependent translocation or activity of PKCδ. Thus, the increased affinity of the C1 domain of PKCδ allows this isozyme to respond to DAG alone, whereas conventional PKC isozymes require the coordinated action of Ca2+ binding to the C2 domain and DAG binding to the C1 domain for activation.
doi:10.1074/jbc.M510251200
PMCID: PMC2913972  PMID: 16293612
21.  Spatiotemporal dynamics of lipid signaling: protein kinase C as a paradigm 
IUBMB life  2008;60(12):782-789.
Summary
The lipid second messenger diacylglycerol (DAG) acutely controls the rate, amplitude, duration, and location of protein kinase C (PKC) activity in the cell. There are three classes of PKC isozymes and, of these, the conventional and novel isozymes are acutely controlled by DAG. The kinetics of DAG production at various intracellular membranes, the intrinsic affinity of specific isoforms for DAG-containing membranes, the coordinated use of additional membrane-binding modules, the intramolecular regulation of DAG sensitivity, and the competition from other DAG-responsive proteins together result in a unique, context-dependent activation signature for each isoform. This review focuses on the spatiotemporal dynamics of PKC activation and how it is controlled by lipid second messengers.
doi:10.1002/iub.122
PMCID: PMC2718795  PMID: 18720411
22.  Discovery of Small Molecule Inhibitors of the PH Domain Leucine-Rich Repeat Protein Phosphatase (PHLPP) by Chemical and Virtual Screening 
Journal of Medicinal Chemistry  2010;53(19):6899-6911.
PH domain Leucine-rich repeat protein phosphatase (PHLPP) directly dephosphorylates and inactivates Akt and protein kinase C, poising it as a prime target for pharmacological intervention of two major survival pathways. Here we report on the discovery of small molecule inhibitors of the phosphatase activity of PHLPP, a member of the PP2C family of phosphatases for which there are no general pharmacological inhibitors. First, the Diversity Set of the NCI was screened for inhibition of the purified phosphatase domain of PHLPP2 in vitro. Second, selected libraries from the open NCI database were docked into a virtual model of the phosphatase domain of PHLPP2, previously trained with our experimental data set, unveiling additional inhibitors. Biochemical and cellular assays resulted in the identification of two structurally diverse compounds that selectively inhibit PHLPP in vitro, increase Akt signaling in cells, and prevent apoptosis. Thus, chemical and virtual screening has resulted in the identification of small molecules that promote Akt signaling by inhibiting its negative regulator PHLPP.
doi:10.1021/jm100331d
PMCID: PMC2951065  PMID: 20836557

Results 1-22 (22)