Lodewijk V. Dekker

Protein kinase C - a question of specificity

Following the initial identification of protein kinase C (PKC) by Nishizuka and co-workers in the late seventies, a wealth of information on this protein kinase has accumulated. Perhaps most striking was the realization that PKC is not just a single polypeptide but in fact consists of a large family of related proteins. These PKC isotypes are unique, not only with respect to primary structure, but also on the basis of expression patterns, subcellular localization, activation in vitro and responsiveness to extra-cellular signals. This review focuses on the heterogeneity within the PKC family and highlights some of the recent evidence that the isotypes might have separate and unique functions in the cell.

Specific Involvement of PKC-ε in Sensitization of the Neuronal Response to Painful Heat

Abstract Pain is unique among sensations in that the perceived intensity increases, or sensitizes, during exposure to a strong stimulus. One important mediator of sensitization is bradykinin (BK), a peptide released as a consequence of tissue damage. BK enhances the membrane ionic current activated by heat in nociceptive neurons, using a pathway that involves activation of protein kinase C (PKC). We find that five PKC isoforms are present in sensory neurons but that only PKC-e is translocated to the cell membrane by BK. The heat response is sensitized when constitutively active PKC-e is incorporated into nociceptive neurons. Conversely, BK-induced sensitization is suppressed by a specific peptide inhibitor of PKC-e. We conclude that PKC-e is principally responsible for sensitization of the heat response in nociceptors by bradykinin.

Critical research gaps and translational priorities for the successful prevention and treatment of breast cancer

IntroductionBreast cancer remains a significant scientific, clinical and societal challenge. This gap analysis has reviewed and critically assessed enduring issues and new challenges emerging from recent research, and proposes strategies for translating solutions into practice.MethodsMore than 100 internationally recognised specialist breast cancer scientists, clinicians and healthcare professionals collaborated to address nine thematic areas: genetics, epigenetics and epidemiology; molecular pathology and cell biology; hormonal influences and endocrine therapy; imaging, detection and screening; current/novel therapies and biomarkers; drug resistance; metastasis, angiogenesis, circulating tumour cells, cancer ‘stem’ cells; risk and prevention; living with and managing breast cancer and its treatment. The groups developed summary papers through an iterative process which, following further appraisal from experts and patients, were melded into this summary account.ResultsThe 10 major gaps identified were: (1) understanding the functions and contextual interactions of genetic and epigenetic changes in normal breast development and during malignant transformation; (2) how to implement sustainable lifestyle changes (diet, exercise and weight) and chemopreventive strategies; (3) the need for tailored screening approaches including clinically actionable tests; (4) enhancing knowledge of molecular drivers behind breast cancer subtypes, progression and metastasis; (5) understanding the molecular mechanisms of tumour heterogeneity, dormancy, de novo or acquired resistance and how to target key nodes in these dynamic processes; (6) developing validated markers for chemosensitivity and radiosensitivity; (7) understanding the optimal duration, sequencing and rational combinations of treatment for improved personalised therapy; (8) validating multimodality imaging biomarkers for minimally invasive diagnosis and monitoring of responses in primary and metastatic disease; (9) developing interventions and support to improve the survivorship experience; (10) a continuing need for clinical material for translational research derived from normal breast, blood, primary, relapsed, metastatic and drug-resistant cancers with expert bioinformatics support to maximise its utility. The proposed infrastructural enablers include enhanced resources to support clinically relevant in vitro and in vivo tumour models; improved access to appropriate, fully annotated clinical samples; extended biomarker discovery, validation and standardisation; and facilitated cross-discipline working.ConclusionsWith resources to conduct further high-quality targeted research focusing on the gaps identified, increased knowledge translating into improved clinical care should be achievable within five years.

Phosphorylation of B-50 (GAP43) is correlated with neurotransmitter release in rat hippocampal slices

Abstract: Recent studies have demonstrated that phorbol diesters enhance the release of various neurotransmitters. It is generally accepted that activation of protein kinase C (PKC) is the mechanism by which phorbol diesters act on neurotransmitter release. The action of PKC in neurotransmitter release is very likely mediated by phosphorylation of substrate proteins localized in the presynaptic nerve terminal. An important presynaptic substrate of PKC is B‐50. To investigate whether B‐50 mediates the actions of PKC in neurotransmitter release, we have studied B‐50 phosphorylation in intact rat hippocampal slices under conditions that stimulate or inhibit PKC and neurotransmitter release. The slices were labelled with [32P]orthophosphate. After treatment, the slices were homogenized, B‐50 was immunoprecipitated from the slice homogenate, and the incorporation of 32P into B‐50 was determined. Chemical depolarization (30 μM K+) and the presence of phorbol diesters, conditions that stimulate neurotransmitter release, separately and in combination, also enhance B‐50 phosphorylation. Polymyxin B, an inhibitor of PKC and neurotransmitter release, decreases concentration dependently the depolarization‐induced stimulation of B‐50 phosphorylation. The effects of depolarization are not detectable at low extracellular Ca2+ concentrations. It is concluded that in rat hippocampal slices B‐50 may mediate the action of PKC in neurotransmitter release.

Lipid rafts determine efficiency of NADPH oxidase activation in neutrophils

We have investigated the contribution of lipid rafts to activation of the NADPH oxidase enzyme system in neutrophils. Membrane‐bound NADPH oxidase subunits are present in the lipid raft compartment of neutrophils. Cytosolic NADPH oxidase components are mainly absent from but are recruited to rafts upon Fcγ receptor activation. In parallel, protein kinase C isotypes are recruited to the rafts. Kinetic analysis of NADPH oxidase activation revealed that rafts determine the onset but not the maximal rate of enzyme activity. Thus lipid rafts serve to physically juxtapose the NADPH oxidase effector, protein kinase C and Fcγ receptor, resulting in efficient coupling.

The protein kinase C and protein kinase C related gene families

Protein kinase C is an important target enzyme for lipid second messengers. Recent developments have focused on the tertiary structure analysis of domains present in protein kinase C and in combination with functional approaches such as mutagenesis and domain expression have generated a detailed understanding of the modular mechanism by which lipids cause activation. This provides a reference for the study of the protein kinase C-related kinases, a recently identified new class of kinases that are highly related to protein kinase C in their catalytic characteristics but have distinct regulatory features.

Activation of PRK1 by Phosphatidylinositol 4,5-Bisphosphate and Phosphatidylinositol 3,4,5-Trisphosphate A COMPARISON WITH PROTEIN KINASE C ISOTYPES

As potential targets for polyphosphoinositides, activation of protein kinase C (PKC) isotypes (β1, ε, ζ, η) and a member of the PKC-related kinase (PRK) family, PRK1, has been compared in vitro. PRK1 is shown to be activated by both phosphatidylinositol 4,5-bisphosphate (PtdIns 4,5-P2) as well as phosphatidylinositol 3,4,5-trisphosphate (PtdIns-3,4,5-P3) either as pure sonicated lipids or in detergent mixed micelles. When presented as sonicated lipids, PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were equipotent in activating PRK1, and, furthermore, sonicated phosphatidylinositol (PtdIns) and phosphatidylserine (PtdSer) were equally effective. In detergent mixed micelles, PtdIns-4,5-P2 and PtdIns-3,4,5-P3 also showed a similar potency, but PtdIns and PtdSer were 10-fold less effective in this assay. Similarly, PKC-β1, -ε, and -η were all activated by PtdIns-4,5-P2 and PtdIns-3,4,5-P3 in detergent mixed micelles. The activation constants for PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were essentially the same for all the kinases tested, implying no specificity in this in vitro analysis. Consistent with this conclusion, the effects of PtdIns-4,5-P2 and PtdIns-3,4,5-P3 were found to be inhibited at 10 mM Mg2+ and mimicked by high concentrations of inositol hexaphosphate and inositol hexasulfate. The similar responses of these two classes of lipid-activated protein kinase to these phosphoinositides are discussed in light of their potential roles as second messengers.

Crystal structure of the C2 domain from protein kinase C-δ

BACKGROUND The protein kinase C (PKC) family of lipid-dependent serine/theonine kinases plays a central role in many intracellular eukaryotic signalling events. Members of the novel (delta, epsilon, eta, theta) subclass of PKC isotypes lack the Ca2+ dependence of the conventional PKC isotypes and have an N-terminal C2 domain, originally defined as V0 (variable domain zero). Biochemical data suggest that this domain serves to translocate novel PKC family members to the plasma membrane and may influence binding of PKC activators. RESULTS The crystal structure of PKC-delta C2 domain indicates an unusual variant of the C2 fold. Structural elements unique to this C2 domain include a helix and a protruding beta hairpin which may contribute basic sequences to a membrane-interaction site. The invariant C2 motif, Pro-X-Trp, where X is any amino acid, forms a short crossover loop, departing radically from its conformation in other C2 structures, and contains a tyrosine phosphorylation site unique to PKC-delta. This loop and two others adopt quite different conformations from the equivalent Ca(2+)-binding loops of phospholipase C-delta and synaptotagmin I, and lack sequences necessary for Ca2+ coordination. CONCLUSIONS The N-terminal sequence of Ca(2+)-independent novel PKCs defines a divergent example of a C2 structure similar to that of phospholipase C-delta. The Ca(2+)-independent regulation of novel PKCs is explained by major structural and sequence differences resulting in three non-functional Ca(2+)-binding loops. The observed structural variation and position of a tyrosine-phosphorylation site suggest the existence of distinct subclasses of C2-like domains which may have evolved distinct functional roles and mechanisms to interact with lipid membranes.

Regulation of a G protein‐gated inwardly rectifying K+ channel by a Ca2+‐independent protein kinase C

1 Members of the Kir3.0 family of inwardly rectifying K+ channels are expressed in neuronal, atrial and endocrine tissues and play key roles in generating late inhibitory postsynaptic potentials (IPSPs), slowing heart rate and modulating hormone release. They are activated directly by Gβγ subunits released in response to Gi/o‐coupled receptor stimulation. However, it is not clear to what extent this process can be dynamically regulated by other cellular signalling systems. In this study we have explored pathways activated by the Gq/11‐coupled M1 and M3 muscarinic receptors and their role in the regulation of Kir3.1+3.2A neuronal‐type channels stably expressed in the human embryonic kidney cell line HEK293. 2 We describe a novel biphasic pattern of behaviour in which currents are initially stimulated but subsequently profoundly inhibited through activation of M1 and M3 receptors. This contrasts with the simple stimulation seen through activation of M2 and M4 receptors. 3 Channel stimulation via M1 but not M3 receptors was sensitive to pertussis toxin whereas channel inhibition through both M1 and M3 receptors was insensitive. In contrast over‐expression of the C‐terminus of phospholipase Cβ1 or a Gq/11‐specific regulator of G protein signalling (RGS2) essentially abolished the inhibitory phase. 4 The inhibitory effects of M1 and M3 receptor stimulation were mimicked by phorbol esters and a synthetic analogue of diacylglycerol but not by the inactive phorbol ester 4αphorbol. Inhibition of the current by a synthetic analogue of diacylglycerol effectively occluded any further inhibition (but not activation) via the M3 receptor. 5 The receptor‐mediated inhibitory phenomena occur with essentially equal magnitude at all intracellular calcium concentrations examined (range, 0‐669 nm). 6 The expression of endogenous protein kinase C (PKC) isoforms in HEK293 cells was examined by immunoblotting, and their translocation in response to phorbol ester treatment by cellular extraction. The results indicated the expression and translocation of the novel PKC isoforms PKCδ and PKCε. 7 We also demonstrate that activation of such a pathway via both receptor‐mediated and receptor‐independent means profoundly attenuated subsequent channel stimulation by Gi/o‐coupled receptors. 8 Our data support a role for a Ca2+‐independent PKC isoform in dynamic channel regulation, such that channel activity can be profoundly reduced by M1 and M3 muscarinic receptor stimulation.

Noradrenaline Release from Streptolysin O‐Permeated Rat Cortical Synaptosomes: Effects of Calcium, Phorbol Esters, Protein Kinase Inhibitors, and Antibodies to the Neuron‐Specific Protein Kinase C Substrate B‐50 (GAP‐43)

Abstract: We studied the molecular mechanism of noradrenaline release from the presynaptic terminal and the involvement of the protein kinase C substrate B‐50 (GAP‐43) in this process. To gain access to the interior of the presynaptic terminal, we searched for conditions to permeate rat brain synaptosomes by the bacterial toxin streptolysin O. A crude synaptosomal/mitochondrial preparation was preloaded with [3H]noradrenaline. After permeation with 0.8 IU/ml streptolysin O, noradrenaline efflux could be induced in a concentration‐dependent manner by elevating the free Ca2+ concentration from 10−8 to 10−5M. Efflux of the cytosolic marker protein lactate dehydrogenase was not affected by this increase in Ca2+. Ca2+‐induced efflux of noradrenaline was largely dependent on the presence of exogenous ATP. Changing the Na+/K+ ratio in the buffer did not affect Ca2+‐induced noradrenaline release. Release of noradrenaline could also be evoked by phorbol esters, indicating the involvement of protein kinase C. Ca2+‐ and phorbol ester‐induced release were not additive at higher phorbol ester concentrations (>10−7M). We compared the sensitivities of Ca2+‐ and phorbol ester‐induced release of noradrenaline to the protein kinase inhibitors H‐7 and polymyxin B and to antibodies raised against synaptic protein kinase C substrate B‐50. Ca2+‐induced release was inhibited by B‐50 antibodies and polymyxin B, but not by H‐7; phorbol ester‐induced release was inhibited by polymyxin B and by H‐7, but only marginally by antibodies to B‐50. We suggest that phorbol esters and Ca2+ stimulate noradrenaline release through different mechanisms and that the essential role of B‐50 in Ca2+‐induced noradrenaline release may involve other properties of B‐50 besides protein kinase C–mediated phosphorylation.