Identification of cortactin as a PKD substrate:potential role in cell migration

Recent werd er voor Proteïne kinase D (PKD) een rol gesuggereerd in celadhesie, celmigratie en celinvasie. Maar de exacte functie van PKD in deze processen of de hierbij betrokken substraten zijn helaas onbekend. Daarom is het doel van dit werk om de fysiologische substraten, die betrokken zijn in deze gebeurtenissen te identificeren en hierdoor PKD een plaats te geven in het uitgebreide web van signaaltransductie.De cadherine familie van transmembraan glycoproteïnen en de geassocieerde eiwitten (zoals alpha-, beta- of gamma-catenin) vormen een zeer belangrijk cellulair systeem dat verantwoordelijk is voor de regulatie van celadhesie en invasie. Omdat er reeds effecten beschreven zijn van PKD in celadhesie en invasie en omdat er bovendien indicaties zijn dat phorbol esters, die PKD kunnen activeren, de functie van E-cadherine regelen, werd het eerste deel van deze thesis gewijd aan het onderzoeken van de rol van PKD in dit E-cadherine/catenine systeem. We slaagden er niet in om aan te tonen dat PKD en E-cadherine of alpha-, beta- en gamma-catenin kunnen intererageren, of dat deze eiwitten door PKD kunnen gefosforyleerd worden. Bovendien zagen we ook geen functioneel effect van PKD op celaggregatie of celinvasie.In het tweede deel van deze thesis werd de mogelijke rol van PKD in celmigratie bestudeerd. Waarbij we voornamelijk streefden naar het vinden van een moleculair verband tussen PKD en celmotiliteit. Aangezien PKD een complex vormt met cortactin en paxillin, twee eiwitten die betrokken zijn in celmigratie, hebben we onderzocht of deze proteïnen PKD substraten zijn. Onze resultaten tonen aan dat PKD paxillin niet kan fosforyleren, maar dat cortactin wel een PKD substraat is.Cortactin is een F-actine bindend eiwit dat netwerken van F-actine kan stabiliseren en dat bovendien door de activering van het Arp2/3 complex bijdraagt tot actine polymerisatie. Met behulp van massa spectrometrie konden we aantonen dat PKD cortactin fosforyleert op Ser 113, gelegen in het eerste cortactin repeat , verder op Ser 298, gelegen in het tweede cortactin repeat, en ook nog op Ser 345 and Ser 348, die juist vóór het helicaal proline rijke domein gelegen zijn. We ontwikkelden fosfospecieke antilichamen tegen deze sites en met behulp van deze antilichamen bestudeerden we de in vivo fosforylatie van cortactin door PKD. Kinetische studies toonden aan dat fosforylatie van Ser 298 verdere fosforylaties op Ser 345 en Ser 348 stimuleert. We zagen ook dat Src, een gekend cortactin kinase, de PKD gemedieerde in vivo fosforylatie stimuleert. Dit effect van Src wordt niet veroorzaakt door prefosforylatie van cortactin, noch door activatie van PKD door Src. De fosforylatie van cortactin door PKD heeft geen effect heeft op de subcellulaire lokalisatie, of op de translokatie van cortactin naar podosomen of membrane ruffles . Maar we konden wel aantonen dat cortactin inderdaad een moleculaire connectie is tussen PKD en celmigratie aangezien PKD gemedieerde cortactin fosforylatie een negatieve invloed heeft op de LPA geïnduceerde celmigratie.status: publishe

From alpha to omega with abeta: targeting the multiple molecular appearances of the pathogenic Peptide in Alzheimer's disease

Amyloid beta (Abeta) is the main component of one of the major pathological hallmarks of Alzheimer's disease and is generally considered as one of the earliest factors that induce the pathogenic cascade. Abeta is produced from a larger precursor protein through proteolytic cleavage by secretase activities, which results in fragments that differ in size depending on the cleavage site used to create the C-terminus. In addition, heterogeneity at the N-terminus is created by proteases/peptidases. Moreover, various amino acid modifications further enhance the heterogeneity of Abeta that accumulates in Alzheimer brain. All these species with their different N-and C termini, with or without modifications have different aggregation properties. Abeta requires an aggregated state to be pathogenic and the exact aggregation state is a major determinant of the cellular effects of Abeta: smaller oligomeric aggregates are more neurotoxic, whereas large fibrillar aggregates are generally more associated with a glial response. It is therefore increasingly clear that Abeta is not a single entity, but a peptide with multiple molecular appearances. In this review we will discuss the mechanisms leading to the generation of the different Abeta species and their involvement in Alzheimer pathogenesis. This will be discussed in the framework of therapeutic approaches that target one of the steps in the biogenesis of toxic Abeta species: inhibition of the formation of Abeta, inhibition of aggregation and stimulation of its degradation or clearanc

Protein kinase D: a family affair

The protein kinase D family of enzymes consists of three isoforms: PKD1/PKCmu PKD2 and PKD3/PKCnu. They all share a similar architecture with regulatory sub-domains that play specific roles in the activation, translocation and function of the enzymes. The PKD enzymes have recently been implicated in very diverse cellular functions, including Golgi organization and plasma membrane directed transport, metastasis, immune responses, apoptosis and cell proliferatio

Characterization of EVL-I as a protein kinase D substrate

EVL-I is a splice variant of EVL (Ena/VASP like protein), whose in vivo function and regulation are still poorly understood. We found that Protein Kinase D (PKD) interacts in vitro and in vivo with EVL-I and phosphorylates EVL-I in a 21 amino acid alternately-included insert in the EVH2 domain. Following knockdown of the capping protein CPbeta and spreading on laminin, phosphorylated EVL-I can support filopodia formation and the phosphorylated EVL-I is localized at filopodial tips. Furthermore, we found that the lamellipodial localization of EVL-I is unaffected by phosphorylation, but that impairment of EVL-I phosphorylation is associated with ruffling of lamellipodia upon PDBu stimulation. Besides the lamellipodial and filopodial localization of phosphorylated EVL-I in fibroblasts, we determined that EVL-I is hyperphosphorylated and localized in the cell-cell contacts of certain breast cancer cells and mouse embryo keratinocytes. Taken together, our results show that phosphorylated EVL-I is present in lamellipodia, filopodia and cell-cell contacts and suggest the existence of signaling pathways that may affect EVL-I via phosphorylation of its EVH2 domai

Disturbed Ca2+ Homeostasis Increases Glutaminyl Cyclase Expression; Connecting Two Early Pathogenic Events in Alzheimer's Disease In Vitro

A major neuropathological hallmark of Alzheimer's disease (AD) is the deposition of aggregated beta amyloid (A beta) peptide in the senile plaques. Ab is a peptide of 38-43 amino acids and its accumulation and aggregation plays a key role early in the disease. A large fraction of beta amyloid is N-terminally truncated rendering a glutamine that can subsequently be cyclized into pyroglutamate (pE). This makes the peptide more resistant to proteases, more prone to aggregation and increases its neurotoxicity. The enzyme glutaminyl cyclase (QC) catalyzes this conversion of glutamine to pE. In brains of AD patients, the expression of QC is increased in the earliest stages of pathology, which may be an important event in the pathogenesis. In this study we aimed to investigate the regulatory mechanism underlying the upregulation of QC expression in AD. Using differentiated SK-N-SH as a neuronal cell model, we found that neither the presence of A beta peptides nor the unfolded protein response, two early events in AD, leads to increased QC levels. In contrast, we demonstrated increased QC mRNA levels and enzyme activity in response to another pathogenic factor in AD, perturbed intracellular Ca2+ homeostasis. The QC promoter contains a putative binding site for the Ca2+ dependent transcription factors c-fos and c-jun. C-fos and c-jun are induced by the same Ca2+-related stimuli as QC and their upregulation precedes QC expression. We show that in the human brain QC is predominantly expressed by neurons. Interestingly, the Ca2+-dependent regulation of both c-fos and QC is not observed in non-neuronal cells. Our results indicate that perturbed Ca2+ homeostasis results in upregulation of QC selectively in neuronal cells via Ca2+-dependent transcription factors. This suggests that disruption of Ca2+ homeostasis may contribute to the formation of the neurotoxic pE A beta peptides in Alzheimer's diseas

An enzyme-linked immunosorbent assay for protein kinase D activity using phosphorylation site-specific antibodies

The protein kinase D (PKD) family is a novel group of kinases that are involved in the regulation of cell proliferation and apoptosis, and several other physiological processes. Hence, these enzymes are attractive targets for pharmacological intervention, but no specific PKD inhibitors are known. With this in mind, we have developed a high-throughput, non-radioactive enzyme-linked immunosorbent assay (ELISA) method to monitor the PKD activity with myelin basic protein (MBP) as substrate. We determined that MBP is phosphorylated by PKD on Ser-160 and that this phosphorylation can be quantified in ELISAs, by the use of phosphorylation site-specific antibodies. Antibodies were developed that are highly specific for the MBP peptide sequence surrounding the phosphorylated Ser-160. We show that our high-throughput kinase assay is useful not only for determining the cellular PKD activity but also to screen for PKD-inhibitory compounds. Our ELISA has advantages over the current radioisotope kinase assay in terms of simplicity and environmental safet

ER Ca²⁺ depletion increases QC levels.

<p>Differentiated SK-N-SH cells were treated with 1 µM TG for 16 h with or without pre-incubation with 5 µM BAPTA-AM (B) for 1 h. Shown are the average + SD of normalized QC mRNA levels of n = 9 from 3 independent experiments. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p≤0.0001).</p

QC mRNA expression is increased in earliest stages of AD pathology.

<p>Expression of QC mRNA was determined in a cohort of patients with varying stages of AD pathology (see materials and methods for details). CON and AD refer to the clinical diagnosis, BS is Braak score for tau pathology, PL is the plaqueload in the hippocampus/entorhinal cortex. Shown is a box-plot of results of the pathological groups as indicated in hippocampus/entorhinal cortex. The expression levels were normalized to eEF2α mRNA. Kruskall-Wallis test was used to evaluate differences between groups followed by the Mann-Whitney U test, to test differences between pairs of groups.</p

Induction of c-fos and c-jun precedes increased QC expression.

<p>Differentiated SK-N-SH cells were treated with 1 µM TG for the times indicated. Shown is the average + SD of normalized QC (A), c-fos (B) and c-jun (C) mRNA levels of triplicates from a representative experiment of three. The expression levels were normalized to eEF2α mRNA, the expression levels in untreated cells are set to 1. Asterisks indicate a significant difference compared to control (*p<0.05; **p≤0.001; ***p≤0.0001).</p