While protein tyrosine kinases (PTKs) have been extensively characterized in eukaryotes, far less is known about their emerging counterparts in prokaryotes. Studies of close to 20 homologs of bacterial protein tyrosine (BY) kinases have inaugurated a blooming new field of research, all since just the end of the last decade. These kinases are key regulators in the polymerization and exportation of the virulence-determining polysaccharides which shield the bacterial from the non-specific defenses of the host. This research is aimed at furthering our understanding of the BY kinases through the use of X-ray crystallography and various in vitro and in vivo experiments. We reported the first crystal structure of a bacterial PTK, the C-terminal kinase domain of E. coli tyrosine kinase (Etk) at 2.5Å resolution. The fold of the Etk kinase domain differs markedly from that of eukaryotic PTKs. Based on the observed structure and supporting evidences, we proposed a unique activation mechanism for BY kinases in Gram-negative bacteria. The phosphorylation of tyrosine residue Y574 at the active site and the specific interaction of P-Y574 with a previously unidentified key arginine residue, R614, unblock the Etk active site and activate the kinase. Both in vitro kinase activity and in vivo antibiotics resistance studies utilizing structure-guided mutants further support the novel activation mechanism. In addition, the level of phosphorylation of their C-terminal Tyr cluster is known to regulate the translocation of extracellular polysaccharides. Our studies have significantly clarified our understanding of how the phosphorylation status on the C-terminal tyrosine cluster of BY kinases affects the oligomerization state of the protein, which is likely the machinery of polysaccharide export regulation. In summary, this research makes a substantial contribution to the rapidly progressing research of bacterial tyrosine kinases. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2008-09-26 12:45:02.924

Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction: Coronary heart disease (CHD) is the leading cause of death worldwide with 3.8 million men and 3.4 million women dying globally each year. Although existing myocardial reperfusion strategies such as thrombolysis and percutaneous coronary intervention (PCI), if applied in a timely manner, limit myocardial infarct size, the mortality and morbidity remains significantly high. Ischaemic preconditioning (IPC) may offer the potential to attenuate myocardial ischaemia/reperfusion injury through cardioprotective signaling pathways which is recruited at the time of myocardial reperfusion, thereby improving clinical outcomes in patients with coronary artery disease. Ischaemic preconditioning is a phenomenon whereby short intermittent episodes of coronary occlusion followed by reperfusion protect the myocardium against a subsequent period of sustained ischaemia. This protection is reflected in the limitation of infarct size and improved functional recovery of the ischaemic heart during reperfusion. Despite intensive research efforts, the promise of an effective cardioprotective strategy using the endogenous protective mechanisms of the heart which underlies IPC, has not yet been materialized. Although progress has been made in terms of signaling mechanisms in the preconditioned heart, the identification of the myocardial reperfusion phase as the critical “window” for cardioprotection, requires the elucidation of the signal transduction pathways during the reperfusion phase after IPC. In view of the above, the aims of the present study were to investigate: i. the involvement of the RISK pathway and p38 MAP kinase pathway in IPC during early and late reperfusion ii. the involvement of heat shock protein-27 (HSP-27), heat shock protein-70 (HSP-70), GSK-3β, CAMKII, AMPK and the transcription factor CREB in the context of IPC during early reperfusion iii. the involvement of autophagy and apoptosis during early and late reperfusion after IPC iv. the correlation of the protein kinases with the hemodynamic parameters of the heart v. the mechanism of IPC by means of two-dimensional (2D) proteomics Methods: The isolated perfused working rat heart model was used with functional recovery as end-point. Hearts were preconditioned (IPC) for 3x5 min global ischaemia, alternated with 5 min reperfusion. Hearts were subjected to 25 min sustained global ischaemia, followed by 5, 10, 15 or 30 min reperfusion when hearts were snap-frozen for western blotting analysis. Alternatively, hearts were reperfused for 30 min to record hemodynamic parameters and measure functional recovery. Non-preconditioned (Non-IPC) hearts were stabilized for 30 min and subjected to 25 min sustained global ischaemia followed by 5, 10, 15 or 30 min reperfusion when hearts were snap-frozen. Alternatively Non-IPC hearts were reperfused for 30 min to serve as control for the 30 min reperfused IPC group. Activation of the protein kinases was determined by western blotting analysis. For the proteomic study mitochondrial and cytosolic proteins were isolated from heart tissue and separated in the first dimension by isoelectric focusing, followed by separation in the second dimension by two dimensional gel electrophoresis. The PD Quest software programme was used to identify significantly expressed protein spots. Protein spots of interest were excised and subjected to in-gel digestion and the resulting peptides were analysed by mass spectrometry. Proteins were identified by Mascot and the Swiss Prot database. Results: Western blotting analysis demonstrated that the RISK pathway and p38 MAPK are activated very early in reperfusion, but the activation is not sustained during the reperfusion period. Autophagy is also upregulated during this early reperfusion phase; it is attenuated in the middle reperfusion phase and increase for a second peak of upregulation in the late reperfusion phase. In addition, we identified CAMKII as a novel marker of functional recovery in IPC after reperfusion. The proteomic analysis identified twenty differentially expressed mitochondrial and thirty six differentially expressed cytosolic proteins between Non-IPC and IPC hearts. Functions ascribed to the majority of these individual proteins were directly related to cardiac metabolism. Conclusion: Activation of the majority of the protein kinases investigated in the present study is associated with the hemodynamic parameters of the heart instead of functional recovery. Results indicated that the variable signaling patterns could be attributed to differences in heart rate and the effect thereof (ejection fraction, minimum and maximum rate of contraction), as a result of sympathetic stimulation due to psychological stress in the animals before slaughtering. Proteomics results demonstrated that IPC hearts which failed after ischaemia /reperfusion are metabolically compromised and “worse off” compared to non-IPC hearts. / AFRIKAANSE OPSOMMING: Inleiding: Koronêre hartsiekte is die vernaamste oorsaak van sterftes wêreldwyd met 3.8 miljoen mans en 3.4 miljoen vrouens wat jaarliks sterf. Alhoewel bestaande miokardiale herperfusie strategieë soos trombolise en perkutane koronêre intervensie (PKI), wanneer betyds toegepas, miokardiale infarktgrootte beperk, bly mortaliteit en morbiditeit steeds hoog. Isgemiese prekondisionering (IPK) beskik oor die potensiaal om miokariale isgemie/herperfusie skade te verminder deur beskermende seinoordragpaaie tydens miokardiale herperfusie te aktiveer en sodoende die pasiënte wat aan koronêre arterie siekte ly, se prognose te verbeter. Isgemiese prekondisionering verwys na die verskynsel waartydens kort episodes van isgemie opgevolg deur herperfusie, die miokardium teen ‘n daaropvolgende langdurige isgemiese insident beskerm. Hierdie beskerming word gereflekteer in die beperking van infarktgrootte en verbeterde funksionele herstel van die isgemiese hart tydens herperfusie. Ten spyte van intensiewe navorsingspogings is die presiese meganisme van endogene beskerming tydens IPK nog nie ten volle ontrafel nie. Die identifisering van die miokardiale herperfusie fase se kritiese “vensterperiode” van beskerming, noodsaak ‘n volledige analise van die seinoordragpaaie wat geaktiveer word tydens die herperfusie fase na IPK. In die lig van bogenoemde, was die doel van die huidige studie om die volgende te ondersoek: i. die betrokkenheid van die RISK seinoordragpad en p38 MAP kinase tydens vroeë en laat herperfusie na IPK ii. die betrokkenheid van “heat shock protein-27” (HSP-27), “heat shock protein- 70” (HSP-70), GSK -3β, CAMKII, AMPK en die transkripsie faktor, CREB, in die konteks van IPK tydens vroeë herperfusie iii. die betrokkenheid van outofagie en apoptose tydens vroeë en laat herperfusie na IPK iv. die korrelasie van die proteïenkinases met die hemodinamiese parameters van die hart v. die meganisme van IPK deur middel van twee dimensionele proteomika Metodes: Die geïsoleerde werkende rothart model, met funksionele herstel as eindpunt, is gebruik. Harte is geprekondisioneer (IPK) met 3x5 min globale isgemie, afgewissel met 5 min herperfusie. Daarna is harte blootgestel aan 25 min volgehoue globale isgemie, gevolg deur 5, 10, 15 of 30 min herperfusie, waartydens harte gevriesklamp is. Alternatiewelik, is harte blootgestel aan 30 min herperfusie ten einde funksionele herstel te meet en hemodinamiese parameters te registreer. Nie-geprekondisioneerde (Non-IPK) harte is gestabiliseer vir 30 min, waarna dit onderwerp is aan 25 min volgehoue globale isgemie, gevolg deur 5, 10, 15 of 30 min herperfusie, waartydens harte gevriesklamp is vir westelike klad analise. Alternatiewelik, is Non-IPK harte onderwerp aan 30 min herperfusie om te dien as kontrole vir die 30 min IPK groep. Aktivering van die proteïenkinases is bepaal deur westelike klad analise. Vir die proteomiese studie, is onderskeidelik mitokondriale en sitosoliese proteïene geïsoleer en geskei in die eerste dimensie met behulp van isoelektriese fokusering, gevolg deur skeiding in die tweede dimensie met behulp van twee dimensionele gel elektroforese. Die PDQuest sagteware program is gebruik om proteïenkolle te identifiseer wat statisties beduidende verskille toon. Proteïenkolle van belang is uitgesny en onderwerp aan in-gel tripsinering en die peptiede wat sodoende verkry is, is deur middel van massa spektrometrie geanaliseer. Proteïene is geïdentifiseer deur Mascot en die Swiss Prot databasis. Resultate: Westelike klad analise het aangetoon dat die RISK pad en p38 MAPK geaktiveer is tydens vroeë herperfusie, maar die aktivering word nie volgehou tydens die hele herperfusie periode nie. Outofagie word gestimuleer tydens die vroeë herperfusie fase; dit word onderdruk in die middel herperfusie fase en bereik ‘n tweede piek van stimulering in die laat herperfusie fase. Die proteomiese analise het onderskeidelik twintig differensieel gereguleerde mitokondriale proteïene en ses en dertig differensieel gereguleerde sitosoliese proteïene geïdentifiseer tussen Non-IPK en IPK. Die grootste persentasie van hierdie proteïene is direk betrokke by miokardiale energie metabolisme. CAMKII is geidentifiseer as ‘n unieke merker van funksionele herstel in IPK tydens reperfusie. Gevolgtrekking: Aktivering van die meeste van die proteïenkinases wat ondersoek is in die huidige studie, is geassosieer met die hemodinamiese parameters van die hart, in plaas van funksionele herstel. Die resultate het aangetoon dat die varierende patrone van kinase aktivering toegeskryf kan word word aan verskille in harttempo en die effek daarvan (ejeksie fraksie, minimum en maksimum tempo van kontraksie), as gevolg van simpatiese stimulasie toegeskryf aan sielkundige stres in die diere voor slagting. Proteomiese analise het getoon dat IPK harte wat faal na isgemie/reperfusie metabolies gekompromiseer is en “slegter daaraan toe” is, in vergelyking met Non-IPK harte.

Src family kinases (SFKs) are key regulators of cell proliferation, differentiation, and survival. The expression of these non-receptor tyrosine kinases is strongly correlated with cancer development and tumor progression. Thus, this family of proteins serves as an attractive drug target. The activation of SFKs can occur via multiple signaling pathways, yet many of them are poorly understood. Here, we summarize the current knowledge on G protein-coupled receptor (GPCR)- mediated regulation of SFKs, which is of considerable interest because GPCRs are among the most widely used pharmaceutical targets. This type of activation can occur through a direct interaction between the two proteins or be allosterically regulated by arrestins and G proteins. We postulate that a rearrangement of binding motifs within the active conformation of arrestin-3 mediates Src regulation by comparison of available crystal structures. Therefore, we hypothesize a potentially different activation mechanism compared to arrestin-2. Furthermore, we discuss the probable direct regulation of SFK by GPCRs and investigate the intracellular domains of exemplary GPCRs with conserved polyproline binding motifs that might serve as scaffolding domains to allow such a direct interaction. Large intracellular domains in GPCRs are often understudied and, in general, not much is known of their contribution to different signaling pathways. The suggested direct interaction between a GPCR and a SFK could allow for a potential immediate allosteric regulation of SFKs by GPCRs and thereby unravel a novel mechanism of SFK signaling. This overview will help to identify new GPCR–SFK interactions, which could serve to explain biological functions or be used to modulate downstream effectors.