Corina E. Antal

Cancer-Associated Protein Kinase C Mutations Reveal Kinase’s Role as Tumor Suppressor

Protein kinase C (PKC) isozymes have remained elusive cancer targets despite the unambiguous tumor promoting function of their potent ligands, phorbol esters, and the prevalence of their mutations. We analyzed 8% of PKC mutations identified in human cancers and found that, surprisingly, most were loss of function and none were activating. Loss-of-function mutations occurred in all PKC subgroups and impeded second-messenger binding, phosphorylation, or catalysis. Correction of a loss-of-function PKCβ mutation by CRISPR-mediated genome editing in a patient-derived colon cancer cell line suppressed anchorage-independent growth and reduced tumor growth in a xenograft model. Hemizygous deletion promoted anchorage-independent growth, revealing that PKCβ is haploinsufficient for tumor suppression. Several mutations were dominant negative, suppressing global PKC signaling output, and bioinformatic analysis suggested that PKC mutations cooperate with co-occurring mutations in cancer drivers. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapies should focus on restoring, not inhibiting, PKC activity.

Intramolecular Conformational Changes Optimize Protein Kinase C Signaling

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 multidomain signal transducer, protein kinase C (PKC), for optimal response to second messengers. This enzyme comprises two diacylglycerol sensors, the C1A and C1B domains, that have a sufficiently high intrinsic affinity for ligand so 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 PKC and that conformational transitions following priming phosphorylations mask the domains so that the lower affinity sensor, the C1B domain, is the primary diacylglycerol binder. The conformational rearrangements of PKC serve as a paradigm for how multimodule transducers optimize their dynamic range of signaling.

Active site inhibitors protect protein kinase C from dephosphorylation and stabilize its mature form

Conformational changes acutely control protein kinase C (PKC). We have previously shown that the autoinhibitory pseudosubstrate must be removed from the active site in order for 1) PKC to be phosphorylated by its upstream kinase phosphoinositide-dependent kinase 1 (PDK-1), 2) the mature enzyme to bind and phosphorylate substrates, and 3) the mature enzyme to be dephosphorylated by phosphatases. Here we show an additional level of conformational control; binding of active site inhibitors locks PKC in a conformation in which the priming phosphorylation sites are resistant to dephosphorylation. Using homogeneously pure PKC, we show that the active site inhibitor Gö 6983 prevents the dephosphorylation by pure protein phosphatase 1 (PP1) or the hydrophobic motif phosphatase, pleckstrin homology domain leucine-rich repeat protein phosphatase (PHLPP). Consistent with results using pure proteins, treatment of cells with the competitive inhibitors Gö 6983 or bisindolylmaleimide I, but not the uncompetitive inhibitor bisindolylmaleimide IV, prevents the dephosphorylation and down-regulation of PKC induced by phorbol esters. Pulse-chase analyses reveal that active site inhibitors do not affect the net rate of priming phosphorylations of PKC; rather, they inhibit the dephosphorylation triggered by phorbol esters. These data provide a molecular explanation for the recent studies showing that active site inhibitors stabilize the phosphorylation state of protein kinases B/Akt and C.

Tuning the signalling output of protein kinase C

Precise control of the amplitude of protein kinase C (PKC) signalling is essential for cellular homoeostasis, and disruption of this control leads to pathophysiological states such as cancer, neurodegeneration and diabetes. For conventional and novel PKC, this amplitude is meticulously tuned by multiple inputs that regulate the amount of enzyme in the cell, its ability to sense its allosteric activator diacylglycerol, and protein scaffolds that co-ordinate access to substrates. Key to regulation of the signalling output of most PKC isoenzymes is the ability of cytosolic enzyme to respond to the membrane-embedded lipid second messenger, diacylglycerol, in a dynamic range that prevents signalling in the absence of agonists but allows efficient activation in response to small changes in diacylglycerol levels. The present review discusses the regulatory inputs that control the spatiotemporal dynamics of PKC signalling, with a focus on conventional and novel PKC isoenzymes.

Gain-of-function mutations in protein kinase Cα (PKCα) may promote synaptic defects in Alzheimer's disease

PKCα variants in some patients with Alzheimer’s disease may mediate the pathological effects of amyloid-β. PKCα variants in Alzheimer’s disease Alzheimer’s disease (AD) is characterized by neurodegeneration and impaired neuronal function in the brain that lead to progressive cognitive loss. The early-onset form of AD is linked to genetic mutations, and accumulation of a protein called amyloid-β occurs in many patients with both early- and late-onset AD. By examining the genetics of a large cohort of families in which late-onset AD was diagnosed, Alfonso et al. found that activating mutations in protein kinase Cα (PKCα) correlated with the disease. Pharmacologically inhibiting PKCα or deleting the gene encoding it prevented amyloid-β from impairing synaptic activity in hippocampal tissue slices from mice. This study suggests that PKCα variants mediate the pathological effects of amyloid-β in some patients with late-onset AD. Alzheimer’s disease (AD) is a progressive dementia disorder characterized by synaptic degeneration and amyloid-β (Aβ) accumulation in the brain. Through whole-genome sequencing of 1345 individuals from 410 families with late-onset AD (LOAD), we identified three highly penetrant variants in PRKCA, the gene that encodes protein kinase Cα (PKCα), in five of the families. All three variants linked with LOAD displayed increased catalytic activity relative to wild-type PKCα as assessed in live-cell imaging experiments using a genetically encoded PKC activity reporter. Deleting PRKCA in mice or adding PKC antagonists to mouse hippocampal slices infected with a virus expressing the Aβ precursor CT100 revealed that PKCα was required for the reduced synaptic activity caused by Aβ. In PRKCA−/− neurons expressing CT100, introduction of PKCα, but not PKCα lacking a PDZ interaction moiety, rescued synaptic depression, suggesting that a scaffolding interaction bringing PKCα to the synapse is required for its mediation of the effects of Aβ. Thus, enhanced PKCα activity may contribute to AD, possibly by mediating the actions of Aβ on synapses. In contrast, reduced PKCα activity is implicated in cancer. Hence, these findings reinforce the importance of maintaining a careful balance in the activity of this enzyme.

Spatiotemporal Dynamics of Phosphorylation in Lipid Second Messenger Signaling

The plasma membrane serves as a dynamic interface that relays information received at the cell surface into the cell. Lipid second messengers coordinate signaling on this platform by recruiting and activating kinases and phosphatases. Specifically, diacylglycerol and phosphatidylinositol 3,4,5-trisphosphate activate protein kinase C and Akt, respectively, which then phosphorylate target proteins to transduce downstream signaling. This review addresses how the spatiotemporal dynamics of protein kinase C and Akt signaling can be monitored using genetically encoded reporters and provides information on how the coordination of signaling at protein scaffolds or membrane microdomains affords fidelity and specificity in phosphorylation events.

Electrostatic and Hydrophobic Interactions Differentially Tune Membrane Binding Kinetics of the C2 Domain of Protein Kinase Cα

Background: The C2 domain is a Ca2+ sensor that drives the first step in the activation of conventional PKC isozymes. Results: Hydrophobic interactions drive membrane association and electrostatic interactions drive membrane retention of the C2 domain. Conclusion: The amplitude and location of conventional PKC signaling is controlled by residues in the C2 domain. Significance: Point mutations in the C2 domain are associated with disease. The cellular activation of conventional protein kinase C (PKC) isozymes is initiated by the binding of their C2 domains to membranes in response to elevations in intracellular Ca2+. Following this C2 domain-mediated membrane recruitment, the C1 domain binds its membrane-embedded ligand diacylglycerol, resulting in activation of PKC. Here we explore the molecular mechanisms by which the C2 domain controls the initial step in the activation of PKC. Using stopped-flow fluorescence spectroscopy to measure association and dissociation rate constants, we show that hydrophobic interactions are the major driving force in the binding of the C2 domain to anionic membranes, whereas electrostatic interactions dominate in membrane retention. Specifically, mutation of select hydrophobic or select basic residues in the Ca2+-binding loops reduces membrane affinity by distinct mechanisms; mutation of hydrophobic residues primarily alters association rate constants, whereas mutation of charged residues affects dissociation rate constants. Live cell imaging reveals that introduction of these mutations into full-length PKCα not only reduces the Ca2+-dependent translocation to plasma membrane but, by impairing the plasma membrane-sensing role of the C2 domain, causes phorbol ester-triggered redistribution of PKCα to other membranes, such as the Golgi. These data underscore the key role of the C2 domain in driving conventional PKC isozymes to the plasma membrane and reveal that not only the amplitude but also the subcellular location of conventional PKC signaling can be tuned by altering the affinity of this module for membranes.

Protein kinase C mechanisms that contribute to cardiac remodelling

Protein phosphorylation is a highly-regulated and reversible process that is precisely controlled by the actions of protein kinases and protein phosphatases. Factors that tip the balance of protein phosphorylation lead to changes in a wide range of cellular responses, including cell proliferation, differentiation and survival. The protein kinase C (PKC) family of serine/threonine kinases sits at nodal points in many signal transduction pathways; PKC enzymes have been the focus of considerable attention since they contribute to both normal physiological responses as well as maladaptive pathological responses that drive a wide range of clinical disorders. This review provides a background on the mechanisms that regulate individual PKC isoenzymes followed by a discussion of recent insights into their role in the pathogenesis of diseases such as cancer. We then provide an overview on the role of individual PKC isoenzymes in the regulation of cardiac contractility and pathophysiological growth responses, with a focus on the PKC-dependent mechanisms that regulate pump function and/or contribute to the pathogenesis of heart failure.

Euphohelioscopin A Is a PKC Activator Capable of Inducing Macrophage Differentiation

To identify small molecules that selectively control hematopoietic stem cell 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 promonocytic effects of this natural product are mediated by PKC activation. In addition to shedding insights into normal hematopoiesis, this work may ultimately facilitate the application of stem cell therapies to a host of myeloid dysfunctions.

Abstract 125: Protein kinase C loss-of-function mutations in cancer reveal role as tumor suppressor

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Protein kinase C (PKC) remains an elusive chemotherapeutic target despite decades of research. To determine whether PKC isozymes function as oncogenes or tumor suppressors, we analyzed 8% of PKC mutations identified in human cancers. Surprisingly, the majority were loss-of-function and none were activating. Loss-of-function mutations were found in all PKC subgroups and acted by impeding 2nd messenger binding or preventing processing phosphorylations. Bioinformatic analysis revealed that PKC mutations might cooperate with co-occurring mutations in cancer drivers. Correction of a patient-identified, loss-of-function PKCβ mutation by CRISPR-mediated genome editing, in a colon cancer cell line, suppressed anchorage-independent growth and reduced tumor growth in xenograft models. Hemizygous deletion provided an anchorage-independent growth advantage, revealing PKC is haploinsufficient for tumor suppression. Several mutations were dominant-negative, suppressing global PKC signaling output. These data establish that PKC isozymes generally function as tumor suppressors, indicating that therapeutic strategies should focus on restoring PKC activity, not inhibiting it. Citation Format: Corina E. Antal, Andrew M. Hudson, Emily Kang, Ciro Zanca, Christopher Wirth, Natalie L. Stephenson, Eleanor W. Trotter, Lisa L. Gallegos, Crispin Miller, Frank Furnari, Tony Hunter, John Brognard, Alexandra C. Newton. Protein kinase C loss-of-function mutations in cancer reveal role as tumor suppressor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 125. doi:10.1158/1538-7445.AM2015-125