The cellular functions of mammalian type II phosphatidylinositol 4-kinases

Type II phosphatidylinositol 4-kinases (PI4KIIs), PI4KIIα and PI4KIIβ, both catalyse phosphatidylinositol 4-phosphate (PI(4)P) synthesis and are implicated in the control of trafficking from the trans-Golgi network (TGN) and endosomal membranes. It has been suggested that these closely related isoforms perform redundant roles. This study addresses the issue of functional overlap, by studying the location of the PI(4)P pools synthesised by each isoform, the associated membrane trafficking routes and the functional consequences of loss of these PI4P pools. The TGN localisations of PI4KIIα and PI4KIIβ could be distinguished by co-immunostaining with TGN markers syntaxin 6 and TGN46, indicating that the isoforms localise to separate TGN domains. In addition, depletion of PI4KII isoforms using small interfering RNA (siRNA) had differential effects on TGN pools of PI(4)P, with PI4KIIα loss significantly affecting a syntaxin 6 positive PI(4)P pool while PI4KIIβ depletion altered a TGN46 positive pool; thus indicating the synthesis of metabolically separate PI(4)P pools by these two isoforms. Depletion of either PI4KII isoform also impaired post-TGN traffic of cation independent mannose 6-phosphate receptor (CI-M6PR) and the endo-lysosomal traffic and degradation of the EGF receptor which is suggestive of overlapping roles for both isoforms in post-TGN traffic. PI4KII gene silencing also had differential effects on the actin cytoskeleton. Loss of PI4KIIα led to increased stress fibre formation while PI4KIIβ depletion induced the formation of functional invadopodia containing membrane type I matrix metalloproteinase (MT1-MMP). This was accompanied by decreased colocalization of MT1-MMP with the endosomal markers Rab5 and Rab7 that control lysosomal trafficking and regulate surface levels of MT1-MMP. However, MT1-MMP showed increased colocalisation with Rab8, which mediates exocytic trafficking and pro-invasive activity of MT1-MMP. In addition, depletion of PI4KIIβ conferred a migratory phenotype on minimally invasive HeLa and MCF-7 cell lines. This cell phenotype was substantiated by oncogenomic database analyses showing that loss of PI4KIIβ expression was a risk factor for numerous human carcinomas

SHP-1 and PDK1 Form a Phosphotyrosine-Dependent Nucleo-Cytoplasmic Shuttling Complex: Implications for Differentiation

SHP-1 is a protein tyrosine phosphatase that often targets the phosphatidylinositol 3'-kinase (PI3K)/Akt signalling pathway. PI3K/Akt signalling regulates cell growth and survival, proliferation and differentiation. Growth factor-stimulated PI3K phospholipid production at the plasma membrane helps to recruit 3'-phosphoinositide-dependent protein kinase-1 (PDK1) and Akt, where PDK1 phosphorylates and activates the pro-survival kinase Akt.Tyrosine phosphorylation of PDK1 may regulate its function and, perhaps more importantly, its nuclear localization. Yet, it is unclear how PDK1 is imported into the nucleus as it does not contain a nuclear localization signal (NLS), although it does contain a nuclear export signal (NES). Interestingly, several tyrosines in PDK1 are targets for Src kinase and are putative target motifs for SHP-1, which does have an NLS.Hypothesis: SHP-1 and PDK1 form a tyrosine-dependent, nucleo-cytoplasmic shuttling complex. Removal of serum from C6 glioma cell cultures induces a platelet-derived growth factor receptor (PDGFR)-sensitive redistribution of PI3K lipid kinase activity to the nucleus. PDK1 tyrosine phosphorylation and its association with SHP-1 are also increased, as is the accumulation of both SHP-1 and PDK1 in the nucleus. Site-directed mutagenesis of tyrosine residues in PDK1 reveals that tyrosine 9 (Tyr9) and Tyr376 are important for the interaction of PDK1 with SHP1, whereas Tyr333 and Tyr 373 are not. Using pharmacological and genetic manipulations, it was demonstrated that SHP-1 and PDK1 shuttle between the nucleus and cytoplasm, and that the C-terminal-expressed NLS of SHP-1 facilitates shuttling, while dephosphorylation of PDK1 Tyr9 and Tyr376 regulates the rate of PDK1 (and by virtue of association, SHP-1) export from the nucleus. The SHP-1/PDK1 complex, which is constitutive in most cell lines, is functionally relevant as indicated by its requirement for NGF-induced differentiation of preneuronal cells to a neuronal phenotype

Chemical Inhibitors of Phosphatidylinositol Transfer Proteins Enable Highly Selective Interference With Specific Pathways of Phosphoinositide Signaling in Cells

Phosphatidylinositol phosphates (PIP) are phosphorylated derivatives of phosphatidylinositol (PtdIns) that signal to and regulate diverse cellular functions including membrane trafficking, cytokinesis, cell cycle regulation and DNA repair. PIP-signaling is regulated by a variety of proteins through degradation, phosphorylation and dephosphorylation. Members of the Sec14-like phosphatidylinositol transfer protein superfamily (Sec14-PITPs) have at least two functions which include lipid-binding platforms and/or `nanoreactors' that direct PtdIns-OH kinase activity to generate discrete PIP-pools. In Chapter 1, I outline the current literature on the Sec14-superfamily and the structurally unrelated START-like PITPs with special emphasis on mammalian PITPs, and how their disruption results in a number of inherited mammalian diseases. Neither Sec14-like or START-like PITPs have been targeted for chemical intervention using small molecule inhibitors (SMIs). The development of PITP-directed SMIs provide applications not only as tool compounds, but also as therapeutic agents that inhibit a number of pathogenic organisms and potentially as activators of defective PITPs. As proof-of-concept, I developed the first PITP-directed SMIs that specifically inhibit the prototype Sec14-like PITP from Saccharomyces cerevisiae. In yeast, Sec14 connects the production of phosphatidylinositol 4-phosphate (PtdIns(4)P) and phosphatidylcholine (PtdCho) metabolism with trafficking through the trans-Golgi/endosomal network. In Chapter 2, I describe the development of the Sec14-directed SMI, 4-chloro-3-nitrophenyl)(4-(2-methoxyphenyl) piperazin-1-yl)methanones or NPPM. These SMIs specifically and directly inhibit Sec14 through its hydrophobic cavity, likely by a halogen-bonding mechanism. Based on my work in Chapter 2, I developed a routine for the rapid validation of novel PITP-directed SMIs from a variety of organisms that will streamline future SMI-identification. Together, these data deliver proof-of-concept that PITP-directed SMIs offer new and generally applicable avenue for intervening with phosphoinositide signaling pathways with selectivities superior to those afforded by contemporary lipid kinase-directed strategies. Finally, the study of PIPs has been advanced through the development of multiple methodologies that both detect and modify PIPs in vivo. In Chapter 3, I discuss current methods used to monitor and manipulate PIP-signaling pathways with special emphasis on SMIs that target PIP-modifying enzymes.Doctor of Philosoph

Quantifying the insulin response in mouse C2C12 skeletal muscle: a minimal modelling approach

Thesis (PhD)--Stellenbosch University, 2021.ENGLISH ABSTRACT: The insulin signalling cascade is one of the most important regulatory and signalling pathways in humans. Dysregulation or dysfunction of the insulin signalling path- ways often underlies the molecular ætiology of diseases such as diabetes, obesity, and Alzheimer’s. In turn, these diseases are the harbingers of various co-morbidities such as cardio-vascular disease, chronic inflammation, and dementia. The healthcare, eco- nomic, personal, and mortality burden of these diseases cannot be overstated. Mathematical modelling of insulin signalling is indispensable in the effort to un- derstand the dynamics of the insulin signalling cascade and how malfunctions therein lead to disease. However, despite the availability and complexity of existing models, few have explicitly connected the signalling cascade, glucose transporter activity, and metabolism with one another. In order to study these interactions, a ‘three-module’ approach was adopted that defined the signalling cascade, glucose transporter activ- ity, and metabolism as core, ‘input-output’ modules. The present work is limited to the signalling cascade and glucose transporter activity modules whereas work by Dr. Cobus van Dyk is concerned with the metabolic module. With this in mind, this thesis sets forth three aims. Firstly, to establish standard- ised culturing conditions which can be used to determine the basal state of insulin signalling and glucose transporter activity. Secondly, to develop a core, mathemati- cal model based on Western blotting and radio-labelled glucose -assay data which is able to describe the concentration- and time-dependence of the signalling cascade and glucose transporter activity in response to insulin. Thirdly, to determine the clustering behaviour of GFP-tagged GLUT4 molecules in response to insulin. The first goal was to standardise culturing conditions. Herein, the ability of high (25mM), medium (15mM), and low (5mM) glucose culturing conditions were evalu- ated with regards to their ability to sensitise or desensitise the insulin signalling cascade as well as the degree to which they are able to induce the differentiation of C2C12 my- oblasts into myocytes. The glucose and lactate concentrations in the external media were used to determine the glucose-lactate flux of the C2C12 cells. This served as a proxy for the induction of insulin-dependent glucose transport and metabolism. A modified Ladd staining protocol was used to assess the degree to which C2C12 cells could differentiate under the culturing protocols. The second goal was to construct a core, mathematical model of insulin signalling and glucose transporter activity. The time-dependent phosphorylation and dephos- phorylation of the insulin receptor and the serine 473 and threonine 308 sites of Akt in response to varying insulin concentrations was investigated using Western blotting techniques. The glucose transporter (GLUT4) activity was assayed using radio-carbon glucose. The data were used to optimise parameters for a core, ODE-based model of the signalling and glucose transporter modules. The third goal, to investigate the clustering behaviour of GLUT4 in response to insulin, was investigated by using confocal microscopy to image GFP-tagged GLUT4 molecules before and after being stimulated with insulin. A hierarchical clustering algorithm as well as further geometric and statistical analyses were used to determine the number, size, density, and distribution of GLUT4 clusters pre and post insulin exposure. Of the remaining chapters, Chapter 1 discusses the background, context, scope, and aims of this study as well as further elaborating on the ‘three module’ approach. The literature review in Chapter 2 provides an overview of the relevant literature as delineated by the scope and aims of this study. The materials and methods are provided in Chapter 3, with specific alterations or methodologies being further discussed in the relevant experimental chapters. The final chapter, Chapter 7, provides the reader with general discussions, limitations, and final thoughts concerning this work.AFRIKAANSE OPSOMMING: Die insulien seinkaskade is een van die belangrikste regulerings- en sein padweë in mense. Disregulering of disfunksie van die insulien sinweë is dikwels onderliggend aan die molekulêre etiologie van siektes soos diabetes, vetsug en Alzheimers. Verder is hierdie siektes die draers van verskillende ko-morbiditeite soos hartvatsiektes, chroniese ontsteking, demensie en ander. Die gesondheids, ekonomiese, persoonlike en sterftes- las van hierdie siektes kan nie oorskat word nie. Wiskundige modellering van insulien seinweë is onontbeerlik in die poging om die dinamika van die insulien seinkaskade te verstaan en hoe wanfunksies daarin tot siektes lei. Ondanks die beskikbaarheid en ingewikkeldheid van die bestaande mod- elle, het min die seinkaskade, glukose-vervoerderaktiwiteit en metabolisme egter ek- splisiet met mekaar verbind. Ten einde hierdie interaksies te bestudeer, is ‘n ‘drie- module’-benadering aangewend wat die seinkaskade, glukose-vervoerderaktiwiteit en metabolisme as kernmodules as ‘n ’inset-uitset’ model gedefinieer het. Die huidige werk is beperk tot die seinkaskade en glukose-vervoerdersaktiwiteitsmodules, terwyl werk deur dr. Cobus van Dyk gemoeid is met die metaboliese module. Met die oog hierop stel hierdie proefskrif drie doelstellings. Eerstens, om ges- tandaardiseerde kweektoestande vas te stel wat gebruik kan word om die basale toe- stand van insulien seine en glukose-vervoerderaktiwiteit te bepaal. Tweedens, om ‘nkern, wiskundige model te ontwikkel gebaseer op Westerse klad-tegnieke en radio- toetsdata, wat die konsentrasie en tydafhanklikheid van die seinkaskade en glukosever- voerder kan beskryf as ‘n gevolg van insulien blootstelling. Derdens, om die groeper- ingsgedrag van GFP-gemerkte GLUT4-molekules in reaksie op insulien te bepaal. Die eerste doelwit, met betrekking tot gestandaardiseerde kweektoestande, word aangebied in hoofstuk 4. Hierin is die vermoë van hoë (25mM), medium (15mM) en lae (5mM) kweektoestande geëvalueer met betrekking tot hul kapasiteit om die insulien seinkaskade te sensitiseer of te desensitiseer, asook die mate waarin hulle die differensiasie van C2C12-myoblaste in miosiete kan veroorsaak. Die skynbare glukose-laktaatvloei in die eksterne media dien as ‘n gevolmagtigde maatstaf vir die induksie van insulienafhanklike glukosevervoer en metabolisme. ‘n Gemodifiseerde LADD-kleuringprotokol is gebruik om die mate waarin C2C12-selle kan onderskei te bepaal. Die tweede doelwit, om ‘n kern, wiskundige model van insulien seinweë en die glukosevervoerder aktiwiteit te konstrueer, word in hoofstuk 5 nagestreef. Die fos- forilering en ontfosforylering van die insulienreseptor en die serien 473 en treonien 308-posisies van die intermediêre seinmolekule (Akt) in reaksie op wisselende in- sulienkonsentrasies, sowel as tyd, is met behulp van Westerse klad-tegnieke onder- soek. Die glukose-vervoerder (GLUT4) -aktiwiteit is met behulp van radio-koolstof glukose ondersoek. Die data is gebruik om parameters te optimaliseer vir ‘n kern- GDV-gebaseerde model van die sein en glukose-vervoermodules. Die derde doelwit, wat die groeperingsgedrag van GLUT4 in reaksie op insulien ondersoek het, word in hoofstuk 6 aangebied. Konfokale mikroskopie is gebruik om GFP-gemerkte GLUT4-molekules wat sonder en met insulien gestimuleer is te analiseer. ‘n Hiërargiese groeperingsalgoritme sowel as verdere meetkundige en statistiese ontledings is gebruik om die aantal, grootte, digtheid en verspreiding van GLUT4- groepe voor en na insulienblootstelling te bepaal. Van die hoofstukke wat nog nie hier bespreek is nie, bied hoofstuk 1 die agter- grond, konteks, omvang en doelstellings van hierdie studie, asook die uitwerking van die ‘drie module’-benadering. Terwyl die literatuuroorsig in hoofstuk 2 bied ‘n onder- soek van die relevante literatuur soos uiteengesit in die omvang en doelstellings van hierdie studie. Die materiaale en metodes word in hoofstuk 3 verskaf, met spesifieke wysigings of metodologieë wat in die betrokke eksperimentele hoofstukke verder be- spreek word. Die finale hoofstuk, hoofstuk 7, sal die leser voorsien met algemene besprekings, beperkings en afsluitende gedagtes rakende hierdie werk.Doctora