PKCγ mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility and kinase activity leading to aberrant MAPK signaling

Verbeek, Dineke S.; Goedhart, Joachim; Bruinsma, Laurie; Sinke, Richard J.; Reits, Eric

doi:10.1242/jcs.027698

Cited by 79 publications

(91 citation statements)

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“…Although neuronal PKC␥ was implicated in the proliferation of astrocytes following repeated morphine administration in vivo, the mechanism of action was not studied (46). In contrast, overexpression of PKC␥ reduced proliferation and resulted in differentiation of lens epithelial cells (47) and glioma cells (48), whereas mutations in PKC␥ reduced its kinase activity, resulting in lower ERK phosphorylation and nuclear ERK translocation, reduced MAPK signaling, and reduced survival of Purkinje cells leading to spinocerebellar ataxia-14 (49). This is the first study demonstrating that 5-HT 2B receptor-mediated proliferation is regulated by PKC␥.…”

Section: Discussionmentioning

confidence: 99%

Protein Kinase Cγ Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Wouters

Roeder

Tharayil

et al. 2009

Journal of Biological Chemistry

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Tight control of cell proliferation is essential to maintain organ size and function. Proliferation needs to be tightly regulated to maintain a critical mass of a particular cell type while preventing dysplasia or malignancy. Cell proliferation is regulated by a complex interaction between extrinsic and intrinsic factors. Extrinsic factors usually signal through cell surface receptors such as various growth factor receptors. 5-Hydroxytryptamine (5-HT, 2 serotonin) is well established as a neurotransmitter and a paracrine factor with over 90% of 5-HT produced by the gastrointestinal tract (1, 2). There is now substantial evidence that, together with these established functions, 5-HT is involved in the control of cell proliferation through various 5-HT receptors, in particular the 5-hydroxytryptamine receptor 2B (5-HT 2B (3-9)). The 5-HT 2B receptor is G q/11 protein-coupled. Activation of the 5-HT 2B receptor regulates cardiac function, smooth muscle contractility, vascular physiology, and mood control. Recently it was demonstrated that activation of the 5-HT 2B receptor also induces proliferation of neurons, retinal cells (3, 4), hepatocytes (5), osteoblasts (8), and interstitial cells of Cajal (ICC) (9). ICC express the 5-HT 2B receptor, and activation by 5-HT induces proliferation of ICC (9). ICC are specialized, mesoderm-derived mesenchymal cells in the gastrointestinal tract. Their best known function is the generation of slow waves (10), but they also conduct and amplify neuronal signals (11, 12), release carbon monoxide to set the intestinal smooth muscle membrane potential gradient (13), and act as mechanosensors (14, 15). Loss of ICC has been associated with pathological conditions such as gastroparesis (16 -18), infantile pyloric stenosis (19, 20), pseudo-obstruction (21, 22), and slow transit constipation (23), whereas increased proliferation of ICC or their precursors is associated with gastrointestinal stromal tumors (24).The mechanisms by which activation of the 5-HT 2B receptor results in increased proliferation are not well understood. In cultured cardiomyocytes, stimulation of the 5-HT 2B receptor activated both phosphatidylinositol 3-kinase (PI3Ј-K)/Akt and ERK1/2/mitogen-activated protein kinase (MAPK) signaling pathways to protect cardiomyocytes from apoptosis (25). On * This work was supported, in whole or in part, by National Institutes of Health Grants DK52766 and DK57061. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental 2 The abbreviations used are: 5-HT, 5-hydroxytryptamine, serotonin; 5-HT 2B , 5-hydroxytryptamine receptor 2B; Gö 6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-a)pyrrolo(

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Section: Discussionmentioning

confidence: 99%

Protein Kinase Cγ Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Wouters

Roeder

Tharayil

et al. 2009

Journal of Biological Chemistry

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“…The mutant displays a higher fluorescence lifetime and hence a reduced FRET efficiency that fits with an open conformation. Detailed methods and statistical analysis are described elsewhere ( Verbeek et al 2008). modified with a small organic fluorophore. In the case of intracellular proteins, these molecules need to be reintroduced into the cell, usually by microinjection.…”

Section: Protein Kinase C Localizationmentioning

confidence: 99%

“…Subsequently, the translocation of both proteins was quantitatively analysed, and the translocation rates were compared. These studies revealed that the mutant PKCs have a substantially higher affinity for phorbol ester, whereas the individual (mutated) domains have less membrane affinity (Verbeek et al 2008). This approach was also used to directly compare the translocation rates of binding domains versus full-length proteins (Raghunath et al 2003).…”

Section: Protein Kinase C Localizationmentioning

confidence: 99%

“…These results demonstrate that a difference in PKC conformation can be measured in cells and stress the importance of using monomeric GFPs. Interestingly, mutants that cause ataxia also showed a decreased FRET signal, demonstrating that the mutants have an altered conformation in living cells (Verbeek et al 2008).…”

Section: Pkc Conformation Sensing With Fretmentioning

confidence: 99%

“…This protein translocates from the membrane to the cytoplasm upon phosphorylation that can be followed in living cells. Also more downstream effectors, such as mitogenactivated protein (MAP) kinases, can be used as indicator of PKC activity (Verbeek et al 2008). …”

Section: Pkc Activity Measured By Fretmentioning

confidence: 99%

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Fluorescence resonance energy transfer imaging of PKC signalling in living cells using genetically encoded fluorescent probes

Goedhart

Gadella

2008

J. R. Soc. Interface.

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Perception of ligands in the extracellular space by transmembrane receptors initiates signal transduction. The conformation change of the receptor induces changes of intracellular signalling components, including altered cellular concentration, altered subcellular location, altered conformation and altered interacting partners. Biochemical approaches have yielded a lot of information about these processes. However, methods that are compatible with analysis of single living cells are often preferred, since cells are highly organized and their response is usually spatially heterogeneous. In addition, the study of signalling cascades requires high temporal resolution. Fluorescence imaging approaches meet these requirements. Moreover, imaging approaches can be combined with genetically encoded green fluorescent protein-based probes that have a high selectivity and sensitivity for the process/molecule of interest. Nowadays, many genetically encoded probes are available for visualizing signalling in living cells. This review is centred on a key regulator of cellular signalling, protein kinase C (PKC). We will discuss imaging approaches that are used for analysing the molecules involved in activation of PKC, visualizing the dynamics of the location of PKC, measuring the conformation of PKC and quantifying the activity of PKC. These approaches are of general interest since they can be applied to study the dynamics, conformation and activity of any protein in living cells.

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Spinocerebellar ataxia type 14: refining clinicogenetic diagnosis in a rare adult‐onset disorder

Schmitz‐Hübsch

Lux

Bauer

et al. 2021

Ann Clin Transl Neurol

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Objectives Genetic variant classification is a challenge in rare adult‐onset disorders as in SCA‐PRKCG (prior spinocerebellar ataxia type 14) with mostly private conventional mutations and nonspecific phenotype. We here propose a refined approach for clinicogenetic diagnosis by including protein modeling and provide for confirmed SCA‐PRKCG a comprehensive phenotype description from a German multi‐center cohort, including standardized 3D MR imaging. Methods This cross‐sectional study prospectively obtained neurological, neuropsychological, and brain imaging data in 33 PRKCG variant carriers. Protein modeling was added as a classification criterion in variants of uncertain significance (VUS). Results Our sample included 25 cases confirmed as SCA‐PRKCG (14 variants, thereof seven novel variants) and eight carriers of variants assigned as VUS (four variants) or benign/likely benign (two variants). Phenotype in SCA‐PRKCG included slowly progressive ataxia (onset at 4–50 years), preceded in some by early‐onset nonprogressive symptoms. Ataxia was often combined with action myoclonus, dystonia, or mild cognitive‐affective disturbance. Inspection of brain MRI revealed nonprogressive cerebellar atrophy. As a novel finding, a previously not described T2 hyperintense dentate nucleus was seen in all SCA‐PRKCG cases but in none of the controls. Interpretation In this largest cohort to date, SCA‐PRKCG was characterized as a slowly progressive cerebellar syndrome with some clinical and imaging features suggestive of a developmental disorder. The observed non‐ataxia movement disorders and cognitive‐affective disturbance may well be attributed to cerebellar pathology. Protein modeling emerged as a valuable diagnostic tool for variant classification and the newly described T2 hyperintense dentate sign could serve as a supportive diagnostic marker of SCA‐PRKCG.

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PKCγ mutations in spinocerebellar ataxia type 14 affect C1 domain accessibility and kinase activity leading to aberrant MAPK signaling

Cited by 79 publications

References 65 publications

Protein Kinase Cγ Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Protein Kinase Cγ Mediates Regulation of Proliferation by the Serotonin 5-Hydroxytryptamine Receptor 2B

Fluorescence resonance energy transfer imaging of PKC signalling in living cells using genetically encoded fluorescent probes

Spinocerebellar ataxia type 14: refining clinicogenetic diagnosis in a rare adult‐onset disorder

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