Les Rémorines (REM) du groupe 1 sont des protéines spécifiques du monde végétale. Malgré leur caractère hydrophile elles sont localisées à la membrane plasmique. La phosphorylation des REM serait potentiellement impliquée dans la signalisation précoce et la défense des végétaux contre les pathogènes. Benschop et al. (2007) détecte AtREM1.3 (Arabidopsis thaliana, groupe 1b) phosphorylée en réponse au traitement par l'éliciteur générale flg22, tandis que Widjaja et al. (2008) a suggéré que la phosphorylation de AtREM1.2 est potentiellement impliquée dans la signalisation précoce à l'infection par Pseudomonas syringae. La fonction précise de la phosphorylation des protéines REM du groupe 1 reste inconnue. Des travaux antérieurs dans le laboratoire ont montré que le mouvement du virus X de la pomme de terre (PVX) est inversement corrélée à l'accumulation de StREM1.3 (Solanum tuberosum) et que StREM1.3 peut interagir physiquement avec la protéine de mouvement TRIPLE GENE BLOC Protein 1 (TGBp1) du PVX (Raffaele et al., 2009). Dans ce travail, nous avons étudié les mécanismes qui sous-tendent les interactions REM-TGBp1 et nous avons essayé de caractériser biochimiquement la kinase qui phosphoryle REM. Les conséquences physiologiques de l'interaction TGBp1 / StREM1.3 et de la phosphorylation de REM en terme de propagation des virus, d’inactivation génique post-transcriptionnelle, de régulation de l’ouverture des plasmodesmes, et d’activation de kinase ont également été étudiés. / The group 1 Remorin (REM) proteins are plant-specific oligomeric proteins that have been reported to localize to the plasma membrane despite their overall hydrophilic nature. There is evidence that the REM protein phosphorylation is potentially implicated in the early signaling and defense. Benschop et al. (2007) detected the AtREM1.3 (Arabidopsis thaliana group 1b of REM protein family) to be phosphorylated in response to treatment with the general elicitor flg22, while the Widjaja et al. (2008) suggested that the phosphorylation of AtREM1.2 is potentially implicated in early signaling upon infection with Pseudomonas syringae. The precise exact function of the group 1 REM protein phosphorylation remains unknown. Previous work in the laboratory showed that Potato virus X (PVX) movement is inversely correlated to potato StREM1.3 accumulation and that StREM1.3 can physically interacts with the movement protein TRIPLE GENE BLOCK PROTEIN 1 (TGBp1) from PVX (Raffaele et al., 2009). In this work, we studied the mechanism underlying the REM-TGBp1 interactions and we tried to characterise biochemically the kinase that phosphorylate REM. The physiological consequences of TGBp1/ StREM1.3 interaction and REM phosphorylation in terms of virus spreading, post-transcriptional gene silencing, plasmodesmata gating, kinase activation were also investigated.

Die Phosphorylierung als posttranslationale Modifikation von Proteinen spielt bei Signalwegen in Zellen eine große Rolle. Für die Entschlüsselung des molekularen Mechanismus der circadianen Uhr ist es daher von Interesse, die Phosphorylierungsstellen beteiligter Proteine zu identifizieren. Im Rahmen dieser Arbeit wurden die Proteine Period I und II mittels Flüssigkeitschromatographie und Tandemmassenspektrometrie (LC-MS/MS) auf Phosphorylierungsstellen untersucht. Hierbei lag der Fokus auf der Verbesserung bestehender Methoden, um eine bessere und umfassendere Identifizierung der Phosphorylierungsstellen, insbesondere in Bezug auf mehrfach phosphorylierte Peptide, zu erreichen. Der Arbeitsablauf beinhaltete die Verwendung mehrerer Proteasen, um eine hohe Sequenzabdeckung des Proteins zu erreichen. Nach der Proteolyse wurden die Phosphopeptide mittels Titandioxid angereichert. Hierbei und bei der LC-MS/MS-Analyse wurde Citrat als Additiv verwendet, welches eine bessere Chromatographie multiphosphorylierter Peptide ermöglicht. Bei der MS/MS-Analyse wurden CID und ETD als Fragmentierungsmethoden eingesetzt. Es konnten durch diese Methodik 30 bzw. 42 Phosphorylierungsstellen an den Proteinen Period I und II identifiziert werden, von denen 26 bzw. 14 zuvor nicht beschrieben waren. Nach der qualitativen Identifizierung wurden quantitative Varianten der optimierten Analytik untersucht, um die biologische Funktion der gefundenen Phosphorylierungsstellen untersuchen zu können. Hierbei wurden das metabolische Labeling der Zellen mit 15N-stickstoffhaltigen Aminosäuren und die säurekatalysierte Isotopenmarkierung auf Peptidebene mit 18O-Sauerstoff untersucht. Mit einer optimierten Variante der säurekatalysierten Isotopenmarkierung mit 18O-Sauerstoff lassen sich die Carboxygruppen der Peptide in 5h 30min mit einer Rate von >97% 18O-Sauerstoff markieren. Mit dieser Methode können weitere funktionelle Untersuchungen der Phosphorylierung an den Period-Proteinen durchgeführt werden. / Protein phosphorylation, a posttranslational modification, plays an important role in signal cascades in cells. In order to understand the molecular mechanism of the circadian clock, it is thus of interest to identify the phosphorylation sites on proteins contributing to the system. During the work for this thesis, the proteins Period I and II were analyzed for phosphorylation sites with liquid chromatography and tandem mass spectrometry (LC-MS/MS). Hereby the focus was on improving existing methods in order to better identify multi-phosphorylated peptides. In the workflow, the Period proteins were digested with several proteases in order to archive a high sequence coverage for analysis. After proteolysis the phosphopeptides were subsequently enriched with titanium dioxide. During phosphopeptide enrichment and reversed phase chromatography, citrate was used as an additive for a better chromatography and recovery of multiphosphorylated peptides. During LC-MS/MS analysis, CID and ETD were used as fragmentation mechanisms in the mass spectrometer. Using these methods, 30 and 42 phosphorylation sites could be identified on the proteins Period I and II, respectively, including 26 and 14 which were previously unpublished. In order to unravel the biological function of these phosphorylation sites, quantitative methods for the optimized LC-MS approach were investigated. This included the metabolic labeling of cells with amino acids containing 15N-nitrogen as well as acid catalyzed 18O-oxygen labeling on peptide level. The developed optimized variant of acid catalyzed 18O-oxygen labeling achieves an inclusion of 18O-oxygen at the peptide carboxy groups with a rate of >97% in 5h 30min. This method can be used for further investigation of the biological function of the phosphorylation on the Period proteins.

La plupart des êtres vivants possèdent une horloge circadienne (période de 24heures). Elle leur permet notamment d’anticiper les changements quotidiens (lumière,température) imposés par la rotation de la terre et d’y adapter leur comportement et leurphysiologie. L’horloge est présente dans la plupart des cellules et repose sur deux boucles derégulation transcriptionnelle négative qui génèrent des oscillations d’ARNm des gènesd’horloge. Un délai entre l’accumulation des ARNm et celle des protéines assure lefonctionnement de la boucle de rétroaction. Ce délai est dû à des modifications posttraductionnellesdes protéines PERIOD et TIMELESS. Les oscillations protéiques sontnotamment contrôlées par leur phosphorylation, l’ubiquitination et la dégradation via leprotéasome. L’ubiquitine ligase SCFSlmb induit la dégradation circadienne de PER et de TIM.SCFJetlag contrôle la dégradation de TIM par la lumière, cette dernière intervenant dans lasynchronisation de l’oscillateur.Au cours de notre étude, nous avons identifié une nouvelle ubiquitine ligase, uncomplexe Cul-3, qui contrôle principalement la stabilité de TIM. Nos résultats indiquent queCul-3 contrôle surtout la stabilité de TIM peu phosphorylé, de façon indépendante de PER,tandis que Slmb contrôle principalement la stabilité de TIM phosphorylé. Nous proposons unmodèle dans l'oscillation de TIM régie par deux systèmes d'ubiquitination: Cul-3 pourretarder l'accumulation nocturne de la protéine, et Slmb pour précipiter sa disparition en finde nuit. / Most living organisms possess a circadian clock (24 hours period). This internal clockallows them to anticipate the daily changes (light, temperature) due to the rotation of theearth and consequently adapt their behavior and physiology. The molecular clock relies ontwo negative feedback loops that generate oscillations of the clock gene mRNA. A delaybetween the accumulation of the mRNAs and the proteins is required for the feedback loop,and is generated by post-translational modifications of PERIOD and TIMELESS. The proteinoscillations are controlled by their phosphorylation, ubiquitination and proteasomedependentdegradation. The ubiquitin ligase SCFSlmb induces the circadian degradation ofPER and TIM. SCFJetlag controls the light-dependent degradation of TIM, which is involved inthe resetting of the clock.In our study, we have identified Cul-3, as a new clock ubiquitin ligase that controlsTIM stability. Our results indicate that Cul-3 mostly controls the stability ofhypophosphorylated TIM, independently of PER, whereas SLMB controls the stability ofphosphorylated TIM. We propose a model where TIM oscillations are regulated by twoubiquitination process. Cul-3 delays the night accumulation of TIM, whereas Slmbprecipitates its degradation at the end of the night.

Crk associated substrate (Cas) is an adaptor protein that becomes phosphorylated upon integrin signaling and influences regulation of cell processes such as migration, proliferation and survival. It consists of multiple domains and regions that can interact with several signaling proteins involved in different signaling pathways. Cas was first discovered as a highly phosphorylated protein in v-Src and v-Crk transformed cells, showing involvement of this protein in cell transformation High level of Breast cancer antiestrogen resistance protein (BCAR-1), a homologue to Cas has shown to correlate with rapid reoccurrence of breast cancer and also create resistance towards Tamoxifen, the widely used medicine for receptor positive breast cancer patients. We have defined boundaries of two regions of Cas termed serine rich region (SRR) and Src binding domain (SBD) respectively and have isolated these segments for biochemical and structural studies. The structure of the serine rich part of Cas has been determined by NMR spectroscopy and reveals a four-helix bundle with unusually long loops. The 14-3-3 protein binds to Cas in a phospho-serine dependent manner and our study suggests that the binding site is located between two helices. The SH2-SH3 domain of a Src family kinase, Lck has also been crystallized in complex with a nine residue long peptide corresponding to the region in Cas that binds to SH2 domains. The structure of this complex has been solved at 2.7Å and shows that Cas binds Src family kinases (SFK) with high affinity suggesting a specific interaction between these two molecules. The biochemical studies on the specific binding site of these molecules show that SFK can bind to any of the phosphorylated tyrosines on the SH2 binding domain of Cas and only one phospho-tyrosine is enough to establish the binding. This binding assay does also indicate that SH3 binding domain of Cas is not essential for SFK binding.

Signal transduction is a crucial event that enables cells to sense and respond to cues from their immediate environment. Guanylyl cyclase C (GC-C) is a member of the family of receptor guanylyl cyclases. GC-C is a single transmembrane protein that responds to its ligands by the production of the second messenger cGMP. The guanylin family of peptides, (including the bacterially produced heat-stable enterotoxin ST) is the ligand for GC-C, elevates intracellular cGMP levels and activates downstream pathways. GC-C regulates the cystic fibrosis transmembrane conductance regulator (CFTR) by inducing phosphorylation by protein kinase G, resulting in chloride ion and fluid efflux. GC-C also regulates cell cycle progression through cGMP-gated Ca2+ channels. These functions are seen in the intestinal epithelium, the primary site for GC-C expression. GC-C as a molecule has been studied in detail, but its functioning in the context of other signaling pathways remains unknown. The aim of the present investigation was to understand the regulation of signal transduction by GC-C and its cross-talk with other signaling pathways operating in the cell. Molecular events that commonly connect components in a signaling pathway are protein phosphorylation and protein-protein interaction. These two aspects are explored in this thesis. The possibility of tyrosine phosphorylation of GC-C has been explored earlier in our laboratory. In vitro studies indicated that the residue Tyr820 was a site for phosphorylation by the Src family of non-receptor tyrosine kinases and those studies also suggested that phosphorylated Tyr820 could bind to the SH2 domain of Src. We generated a nonphosphorylatable mutant of GC-C, GC-CY820F, and a phosphomimetic mutant GC-CY820E to study the effect of phosphorylation of Tyr820, on the functioning of GC-C. A stable cell line of HEK293:GC-CY820F cells was generated and compared with HEK293:GC-CWT. Dose response to ST in the two cell lines showed that cGMP accumulation by GC-CY820F was greater than that of GC-CWT, although the EC50 remained unchanged. The phosphomimetic GC-CY820E mutant receptor was non-responsive to ST. Further in HEK293 cells, phosphorylation of GC-CWT by constitutively active v-Src resulted in decreased ST stimulation and this effect of v-Src was reduced with GC-CY820F. Inhibition of ST stimulation brought about by v-Src required catalytically active Src, as the kinase inactive v-SrcK295R did not inhibit ST stimulation. These results were corroborated by in vitro studies by using the recombinant catalytic domain of GC-C expressed in insect cells and by phosphorylation using a purified kinase, Hck. Observations suggested that phosphorylation of Tyr820 in the catalytic domain of GC-C compromises the guanylyl cyclase activity of GC-C. T84 and Caco-2 colon carcinoma cells endogenously express GC-C. The effect of tyrosine phosphorylation of GC-C was studied by using HgCl2, a known activator of Src kinases, and by the inhibition of protein tyrosine phosphatases using pervanadate, an irreversible inhibitor. Both these ways of achieving increased tyrosine phosphorylation resulted in decreased ST-stimulated cGMP production by GC-C, as suggested from v-Src transfection studies. This decrease was reversed by using a Src kinase specific inhibitor PP2, confirming the role of Src kinases in the inhibition of GC-C activity. Interestingly, in Caco-2 cells that differentiate in culture, the effect of pervanadate on the inhibition of ST-stimulated GC-C activation was dependent on the differentiation stage. Crypt-like cells showed higher inhibition with pervanadate. As they matured into villus-like cells, the effect of pervanadate on GC-C activation was gradually lost. This effect also correlated with a decrease in the expression of Lck, suggesting that in the context of the intestine there could be differential regulation of tyrosine phosphorylation of GC-C along the crypt-villus axis. Intestinal ligated loop assays in rats demonstrated that ST-induced fluid accumulation in the intestine was abrogated on pervanadate treatment. Reduction in this fluid accumulation by pervanadate was not observed with 8-Br-cGMP, a cell permeable analogue of cGMP. This indicated that tyrosine phosphorylation of proteins is important for ST-induced fluid accumulation, and perhaps pervanadate modulates this by phosphorylation of GC-C, thereby causing a reduction in fluid accumulation. Earlier in vitro studies on Src-SH2 binding from the laboratory had suggested the possibility of activation of Src family kinases by GC-C. The activation status of Src kinases was monitored by using phosphorylation-state specific antibody, pSFK416. ST stimulation in T84 cells increased Tyr416 phosphorylation of Src kinases in a time dependent manner, indicating that Src kinases are activated downstream of GC-C. This activation of Src kinases was also seen with the endogenous ligand of GC-C, uroguanylin. Interestingly, 8-Br-cGMP a cell permeable analogue of cGMP that is known to mimic other cellular effects of GC-C, namely Cl-secretion and cell cycle progression, did not activate Src kinases, suggesting that the mechanism of Src kinase activation by GC-C could be independent of cGMP. Binding affinities of Src, Lck, Fyn and Yes SH2 domains to Tyr820 phosphorylated GCC peptide were in the nM range, indicating a high affinity of interaction. In vitro GST-SH2 pull down experiments suggested that phosphorylation of Tyr820 in full length GC-C allows interaction of GC-C to the SH2 domain of Src. These studies suggest a dual cross-talk between Src kinases and GC-C; Src phosphorylation inhibits GC-C signaling and stimulation of GC-C by its ligands activates Src kinases. Interaction of proteins containing SH2 and SH3 domains are commonly found in signaling molecules. In accordance with the observation that there are three PXXP motifs in GCC, many SH3 domains could interact with GC-C. GC-C appears to show a preference to bind the SH3 domains of Fyn, Hck, Abl tyrosine kinases, Grb2 and Crk adaptor proteins, the α-subunit of P85 PI3 kinase, PLC-γ and cortactin to various extents. The SH3 domains of spectrin and Nck did not show any detectable interaction with GC-C. In SH3 pull-down assays, the N-terminal SH3 domain of Crk, CrkSH3 (N), bound GC-C maximally, suggesting that Crk is a good candidate for interaction with GC-C. By overlay analysis, the region of GC-C that binds CrkSH3 (N) was narrowed down to the catalytic domain of GC-C containing a ‘PGLP’ motif. Mutations were generated in GC-C at this site to generate GC-CP916Q and GC-CW918R. These mutations compromised the binding of full length receptor to CrkSH3 (N). In cells, CrkII and GC-C co-transfection inhibited the ST stimulation of GC-C. A CrkII mutant, that has compromised binding through its SH3 domain, did not inhibit the activity of GC-C. CrkII from T84 cells co-immunoprecipitated with GC-C and interestingly, the phosphorylated form of CrkII did not, indicating that GC-C - Crk interaction could be regulated by the phosphorylation of Crk. In summary, this study places GC-C, in the context of tyrosine kinase signaling pathway and interaction with the adaptor protein Crk. These studies suggest that GC-C signal transduction can be altered by cross-talk with other signaling events in the cell. Reversible phosphorylation of tyrosine residues inhibits the activity of GC-C, and this is mediated by Src family kinases. Src kinases themselves are activated on stimulation of GC-C by its ligands, possibly because of SH2 domain interaction with GC-C. Association of Crk by its SH3 domain regulates GC-C functioning primarily by inhibiting ST-stimulated cGMP production. This opens up the possibility of GC-C signaling through a multimeric complex involving other binding partners of Crk, and these cross-talks involving GC-C with the two proto-oncogenes, Src and Crk, might have far reaching consequences in the regulation of cellular functions.

Über die Proteinphosphorylierung in den Mitochondrien der Hefe Saccharomyces cerevisiae ist im Gegensatz zu anderen Kompartimenten nur wenig bekannt. Insbesondere hinsichtlich der physiologischen Bedeutung sowie den an der Modifikation beteiligten Enzymen sind kaum Daten verfügbar. Vor diesem Hintergrund stand die Identifizierung und molekularbiologische Charakterisierung mitochondrialer Proteinkinasen (PKasen) und Proteinphosphatasen (PPasen) im Fokus dieser Arbeit. Unter Verwendung komparativer 2D DIGE-Analysen konnten zwei Strategien erfolgreich verfolgt werden: Zum einen wurde die Konsequenz einer Gendeletion von ausgewählten PKasen bzw. PPasen mit putativer mitochondrialer Lokalisation untersucht. Dabei gelang es, die an der in vivo Regulation des Pyruvatdehydrogenase(PDH)-Komplexes beteiligten Enzyme erstmalig zu identifizieren und im Folgenden deren regulatorisches Zusammenspiel umfassend zu analysieren. Zum anderen wurde in einem inversen Ansatz beispielhaft für die PKase Sat4p untersucht, welche Auswirkungen eine Überexpression dieser Kinase auf das mt Proteom hat. Erste Hinweise, welche zur Identifizierung der PDH-Kinasen (Pkp1p und Pkp2p) bzw. PDH Phosphatasen (Ppp1p und Ppp2p) führten, lieferten die signifikanten Spotänderungen von Pda1p (E1α-Untereinheit des PDH-Komplexes) in den 2D-DIGE-Analysen. Im Folgenden wurde die mitochondriale Lokalisation der vier regulatorischen Enzyme unter Verwendung Epitop-getaggter Varianten nachgewiesen sowie Pda1p in unabhängigen phosphospezifischen Analysen als Target der Phosphorylierung verifiziert. Die Phosphorylierungsstelle von Pda1p konnte massenspektrometrisch dem Serin313 zugeordnet werden. PDH-Aktivitätsmessungen zeigten, dass die Phosphorylierung von Pda1p den PDH Komplex inaktiviert, während eine Dephosphorylierung zur Aktivierung führt. Dabei war der Einfluss der Deletion der PDH Kinasen bzw. der PDH-Phosphatasen unterschiedlich stark ausgeprägt. Während Ppp1p und Ppp2p partiell redundante Funktionen besitzen, lassen die Analysen komplementäre Aktivitäten von Pkp1p und Pkp2p vermuten. Eine physikalische Interaktion der beiden Kinasen wurde in vivo nachgewiesen und deutet auf die Bildung funktioneller Heteromere hin. Durch Analysen in der 2D-BN/SDS-PAGE konnte eine Assoziation der PDH-Kinasen sowie PDH-Phosphatasen mit dem hochmolekularen, etwa 8 MDa großen PDH-Komplex sowie mit PDH-Subkomplexen geringeren Molekulargewichts gezeigt werden. Die Erkenntnisse dieser Arbeit ermöglichten in Verbindung mit denen eigener Vorarbeiten die Erstellung eines Modells zur PDH-Regulation in Saccharomyces cerevisiae. Neben der Aktivitätsregulation durch die von Pkp1p/Pkp2p bzw. Ppp1p/Ppp2p katalysierte Phosphorylierung wird eine Funktion der regulatorischen Enzyme – insbesondere der PDH-Kinasen – an der Assemblierung bzw. Stabilisierung des PDH-Komplexes postuliert. Es konnte somit gezeigt werden, dass in der Hefe ein ähnlicher, aber nicht identischer Regulationsmechanismus vorliegt wie in höheren Eukaryoten. Die zweite Strategie, welche in dieser Arbeit exemplarisch für eine PKase verfolgt wurde, führte zur Identifikation einer bislang unbekannten Funktion der Kinase Sat4p in den Mitochondrien. Es konnte gezeigt werden, dass Sat4p dual lokalisiert in der cytoplasmatischen sowie mitochondrialen Fraktion vorliegt und selbst Target der Phosphorylierung ist. Die Überexpression von Sat4p führte nicht nur zu einem verminderten Wachstum auf nicht fermentierbaren Medien, sondern auch zur Beeinflussung spezifischer mitochondrialer Proteingruppen. Während die Spots der Proteine Pil1p und Lsp1p eine höhere Intensität zeigten, wiesen die Fe/S-Proteine Aco1p und Lys4p eine verminderte „steady-state“-Konzentration auf. Darüber hinaus lagen die Proteine, welche Liponsäure als prosthetische Gruppe tragen (Lat1p, Kgd2p und Gcv3p), im Tet-Sat4-Stamm vorwiegend in ihrer nicht-lipoylierten Form vor. Die Lipoylierungsstellen aller drei Proteine konnten im Wildtyp unter Nutzung von nanoLC-MS/MS erstmals experimentell bestimmt und Lys75 (Lat1p), Lys114 (Kgd2p) bzw. Lys102 (Gcv3p) zugeordnet werden. Die fehlende Lipoylierung der Proteine bzw. die verminderte Proteinkonzentration der Aconitase führte zu einer stark verminderten Aktivität der betroffenen Enzymkomplexe. Neben den in der Literatur beschriebenen putativen Funktionen von Sat4p bei der Regulation cytoplasmatischer Proteine wurde basierend auf den Erkenntnissen der Analysen eine spezifische Funktion der Kinase in den Mitochondrien postuliert. Das Modell schlägt eine Rolle von Sat4p in den späten Schritten der Maturation einer spezifischen Gruppe von mitochondrialer Fe/S-Proteinen vor. Die Beeinträchtigung der Lipoatsynthase Lip5p, welche neben Aco1p und Lys4p ebenfalls zu dieser Gruppe gehört, führt vermutlich sekundär zum beobachteten Verlust der Lipoylierung von Lat1p, Kgd2p und Gcv3p.

A cell is a highly complex, ordered, and above all, a robust system. It copes with in-trennel and external uncertainties like heterogeneous stimuli, errors in processing and execution, and changes within and outside the cell. Maintenance of such a system critically depends on a large body of signalling networks and associated regulatory mechanisms. Of the recurrent manoeuvres in cell signalling, protein phosphorylation is the most prominent, and is used as a switch to transmit information and effect-ate various outcomes. It is estimated that 30% of the entire proteome of a typical eukaryotic cell is phosphorylated at one time or another, almost exclusively at the hydroxyl groups of one or more Seer(S)/Thru(T)/Tyr(Y) residues. This phosphorylation is accomplished through the transfer of g-phosphate of ATP in the presence of cations by a superfamily of enzymes called protein kinases, or STY kinases. In accordance with widespread phosphorylation events, STY kinases form a large and diverse superfamily, constituting 2% of the proteins encoded in an eukaryotic genome and about 500 proteins in the human proteome. Distantly related STY kinases share less than 20% sequence identity, phosphorylate specific substrates, bind to dis-tint interaction partners, localise in different cellular compartments and are regulated by different mechanisms. Despite flexibly accommodating these specific attributes, all STY kinases share a conserved 3-dimensional fold and retain the catalytic function. Moreover, all STY kinases can be manipulated by the signalling machinery to be in the “on” (functionally active) or “off” (inactive) state, thereby adding another layer of regulatory control. Such versatility of the STY kinase domain in harbouring specific substrate recognition motifs, binding interfaces, domain architectures and functional states makes it one of the most influential players in cell signalling and a desirable drug target. Despite decades of studies, a comprehensive understanding of the kinase domain, and the features that dictate its catalytic activity and specificity is lacking. This is reflected by the fact that whereas kinases specifically bind and phosphorylate their cognate substrates, most drugs targeted at them are non-specific and beget cross-reactivity. This gap in understanding potentially ensues from an awry outlook of STY kinases from the viewpoint of sequences and structures alone. It is now well established that the function and regulation of a protein molecule, along with its stability and evolution, is closely related to its dynamics. In this premise, this thesis explores the mechanistic and dynamics underpinnings of STY kinases, and interprets them in the light of their multitude of functional responsibilities and specificities In Chapter 1 of the thesis, we broadly discuss the complexity of cell signalling and the pivotal role of STY kinases in it. After a brief introduction to cell signalling in eukaryotes, several signal cascades mediated by different secondary messengers (camp, cGMP, DAG, IP3) are described. In these signal pathways, modularity is identified as a recurrent theme at all levels of hierarchy: within domain, within protein, within signalling pathway and across signalling pathways. One such modular regulation, protein phosphorylation, is discussed in detail and its catalytic enzyme STY kinase is introduced. An overview and historical perspective of the STY kinase superfamily is presented along with the review of literature pertaining to their sequence, structure and catalytic function characteristics. We note that in the active state, all STY kinases adopt a specific spatial conformation characterised by precise positioning of crucial structural motifs, while the inactive state is usually a case of some deviation from these structural constraints. Chapter 2 addresses a fundamental question in the protein dynamics and function paradigm. If mobility and dynamics of a protein is intimately coupled to its function, how does it manifest in STY kinases? Is there a discernible inter-relationship be-tween the mobility of an STY kinase and its functional competence? To answer these questions, we collated 55 crystal structures of 14 STY kinases from diverse groups and families, and subjected their kinase catalytic domains to Gaussian network model (GNM) based normal mode analysis (NMA). GNM models the kinase structure as a 3-dimensional mass-spring system in a coarse-grained fashion, with masses/nodes at Ca atom positions. Proximate Ca nodes, within a 7 A˚ distance cut-off, are con-nested by identical virtual springs, resulting in a simplified network of Ca-Ca bonded and non-bonded interactions modelled as harmonic potentials. Based purely on the topology of mechanical constraints imposed by the springs, GNM analytically deter-mines the isotropic vibrational normal modes available to the kinase structure. This method approximates the energy of the protein structure harmonically, and thus any micro-motion of the kinase can be theoretically described by a linear cSombination of the calculated normal modes. It is known from previous studies that the modes of low frequencies correspond to biologically feasible and meaningful motions like hinge movements, protein folding and catalysis. We note that the multiple crystal structures analysed in each of the 14 STY kinases are identical in sequence and gross structural fold, and vary only in local backbone conformations corresponding to the functional state of the kinase (active/inactive). Upon examining the fluctuations of kinases in the normal mode of the least frequency (or, global mode), we found systematically higher structural fluctuations in the inactive states when compared to the corresponding active states. This observation held true within individual kinases and across all the 14 kinases. Taken together, a more number of residues have higher fluctuations in the inactive states (n = 1095), than those with higher fluctuations in the active states (n = 525; Chi-square test, p value < 0.05). This skewed fluctuation distribution is in corroboration with higher B-factors and con-formational energies of the inactive state crystal structures. Moreover, high fluctuation is observed across the different inactive forms, except a small fraction of DFG-“in” in-active conformations. Interestingly, the regions of differential fluctuation localised to activation loop, catalytic loop, aC-helix and aG-helix, which are implied in kinase function and regulation. Further investigation of 476 crystal structures of kinase com-plexes with other proteins revealed a remarkable correspondence of these regions of differential fluctuation to contact interfaces. We further verified that this differential fluctuation is not a trivial consequence of bound small molecules or mutations, but an inherent attribute of the kinase catalytic domains. In Chapter 3, we verified the accuracy of differential fluctuation observed between the active and inactive STY kinases, as perceived from GNM based NMA, using the more rigorous method of molecular dynamics (MD) simulations. GNM is minimal-is tic in that the STY kinase catalytic domain is coarse-grained and reduced to a 3-dimensional mechanical network of Ca atom nodes. Thus, the role of side chains and their biophysical character, intra-protein interactions, mutations and bound factors are grossly overlooked. In this premise, we conducted all-atom MD simulations using AMBER ff14SB force-field of 6 structural variants of cAMP-dependent protein kinase (PKA) for 1 ms each. We chose 2 crystal structures of active and inactive PKA (PDB IDs 3FJQ and 1SYK respectively) whose kinase domains shared high structural similarity (gRMSD = 2.6 A)˚. They were modified in silico to obtain 6 starting structures for MD simulations: phosphorylated kinase domain in active and inactive states, kinase do-main along with its C-terminal tail in active and inactive states, active kinase domain bound to ATP/2Mg2+, and unphosphorylated inactive kinase domain. In the absence of external domains, the inactive kinase domain conformation elicits higher mobility in terms of Ca RMSD and Ca RMSF than the active kinase domain. Of the 255 residues in PKA, remarkable 198 residues have higher Ca RMSF in the inactive state, with predominant contributions from ATP binding loop, catalytic loop and aG-helix. In the presence of C-terminal tail, the differential mobility of the kinase domain is exaggerated, with 241 out of 255 residues showing higher Ca RMSF in the inactive state. Upon close investigation, we found that in the presence of C-terminal tail, al-though the mobility of residues is generally suppressed in both the functional states, a few functional regions like activation loop and hinge residues experience higher Ca RMSF in the inactive state. This sheds light on the role of C-terminal tail in the dynam-ics of the activation loop, potentially operating through the hinge residues. Absence of phosphorylation in the inactive kinase domain increases the mobility of residues in general, except of those in the aG-helix. When bound to ATP/2Mg2+, active ki-nase domain (active-holo) showed higher mobility than the active-apo and inactive structures, contrary to the previous results and studies. Intrigued, we examined the simulation closely and found a transition of the active-holo structure to another con-formation, named s2, at 450 ns. Upon analysis of the trajectory before the transition, the active-holo form was indeed found to be more stable and less mobile than the inac-tive state(s). Thus, all the inactive variants are found to be consistently more agile and mobile than their active counterparts, in agreement with the results obtained using NMA. Chapter 4 discusses the transition of the active-holo simulation to a new state, named s2, characterises its structural features and explores the possibility of its func-tional relevance. In the previous chapter, while attempting to verify the presence of differential mobility between various active and inactive forms of PKA through MD simulations, we chanced upon the transition of an active PKA state bound to ATP/2Mg2+ (active-holo) to s2 conformation. The s2 state has a Ca RMSD of up to 4.1 A˚ from the initial starting conformations, mainly contributed by the ATP binding loop, abs-helix, act-helix and age-helix, which are implicated in catalysis and substrate recognition. Once formed, s2 was stable and did not revert back to the active-hole starting structure or any other conformation. We calculated all-vs.-all Ca RMSDs of the conformations sampled during the simulation and identified 3 time periods: 0 - 200 ns of initial conformations similar to the starting structure, 201 - 500 ns of transition, and 501 - 1000 ns of s2 conformations. Principle component analysis (PCA) of the Ca spatial positions during the entire trajectory also categorically exposed two energy wells corresponding to the initial and s2 conformations in the first and second PCs (variance = 56%). Upon systematically comparing the conformers sampled in MD with every known kinase structure, no structure hit with Ca RMSD 2 A˚ was found for conformers sampled after 500 ns, deeming s2 as a novel and hitherto unknown conformation. Investigation of persistent intra-protein interactions unique to the s2 state revealed two stabilising interactions: a salt bridge between K73 and E106 in the b-sheet behind the ATP binding cleft and a network of hydrophobic interactions anchoring act-helix to the age-helix. Aside from these defining interactions, s2 is also characterised by a higher density of intra-protein hydrogen bond network, which stabilises it further. PCA of the MD trajectory indicates the transition of active-hole to s2 to be a process with at least 2 steps, the first being the salt bridge formation. Evolutionary conservation analysis shows that the crucial residues involved in the s2-specific interactions are not reliably conserved across PKAs of other organisms. However, convergence to s2 may still be feasible through other courses of stabilising interactions. From a functional perspective, the s2 conformation opens up the age-helix away from the kinase core and mildly rearranges the catalytic cleft, thereby unmasking a hotspot for sub-strata binding that was absent in the initial structure. In an attempt to replicate the s2 conformation, we performed 4 repeat simulations of the same active-hole starting structure for 1 ms each. Although two of these independent simulations achieved the K73-E106 salt bridge, none of them cloned the complete extent of transition and con-mergence to s2. Instead, we sampled another set of novel conformations, s3, in one of the repeat simulations indicating a disposition for the ATP bound PKA to sample different conformations. Comparative analysis suggests a potential role of C-terminal tail in stabilising the active-hole conformation in physiological conditions. Chapter 5 characterises the extent of conservation of structural fluctuations in ho-mologous STY kinases and interprets the observations in the light of their regulatory diversity. Upon establishing that structural fluctuations of STY kinases carry activity-specific information (Chapter 2) and affirming the same using MD simulations (Chap-ter 3), we hypothesised that the mobility of STY kinases is an important consider-action to understand the basis of their regulatory features as well. In that case, one would expect the structural fluctuations to be better conserved in closely related STY kinases than distantly related ones. To test our hypothesis, we collated 73 crystal structures containing an STY kinase domain in the active conformation and subjected them to GNM based NMA as described above. The global mode structural fluctuations of pairs of STY kinases of varying evolutionary divergence (same-protein, within-subfamily, within-family, within-group and across-groups) were analysed. We found that the closely related STY kinase pairs (of same-protein and within-subfamily cate-goriest) have more conserved and better correlated structural fluctuations than those that were distantly related (of within-group and across-group categories). This con-serration of flexibility did not trivially follow from sequence/structure conservation, since a substantial 65.4% of variation in fluctuations was not accounted by variations in sequences and/or structures. Across the 73 active STY kinases belonging to different groups, we identified a conserved flexibility signature defined by low magnitude fluctuations of residues in and around the catalytic loop. Interestingly, we also identified sub-structural residue-specific fluctuation profiles characteristic of kinases of different categories. Specifically, fluctuation patterns that are statistically unique to kinase groups (AGC, TK) and families (PKA, CDK) were recognised. These fluctuation signatures localise in sites known to participate in protein-protein interactions typical of the kinase group and family concerned. Thus, we report for the first time that residues characterised by fluctuations that are differentially conserved within a group/family are involved in interactions specific to the group/family. Upon the validation of structural fluctuation as an indicative tool to understand kinase-specific interactions, we elucidate an application of this understanding. In SC kinase, we identified local regions around the age-helix to be exhibiting conserved differential fluctuations in comparison to its close relatives EGFR and Abl. Following from the learning that specific fluctuations are correlated with specific binding, we propose this as an attractive target for drug design, with minimal cross-reactivity. Overall, this chapter demonstrates the conservation of fluctuation in STY kinases and underscores the importance of consideration of fluctuations, over and above sequence and structural features, in understanding the roles of sites characteristic of kinases. Chapter 6 documents the frequency of substitution of structural fluctuations in STY kinases over the course of divergent evolution. So far, we had established that structural fluctuations are evidently distinct in the varied functional states assumed by a single STY kinase (Chapter 2-3). In addition, fluctuations are differentially conserved within closely related kinases, but systematically vary across families (Chapter 5). In this chapter, we quantify the structural fluctuation variations in all residues of STY kinases put together. In a sense, this is the fluctuation space available for STY kinases across their functional states, binding modes, and regulatory mechanisms. To accomplish this, we systematically compiled all known eukaryotic kinase domain structures solved at resolutions better than 3 A˚. These structures were then divided into wild-type (harbouring no mutations and having typical amino acids at critical functional sites), pseudo-kinase (harbouring no mutations, but having unconventional amino acids at critical functional sites), disease mutant (harbouring mutations that have imp-plications in diseases) and mutant of unknown effect (harbouring mutations whose physiological manifestation is unknown) categories. Global mode structural fluctuations were determined for every kinase catalytic domain structure in each of the 4 enlisted categories. Similar to Benioff and Benioff’s BLOSUM that summarised the probability of all possible amino acid substitutions in homologous proteins, we documented a ma tricks of fluctuation substitution frequency in the conserved regions of wild-type kinases (named FLOSUM). We observe a positive correlation between the mean and variance of structural fluctuations at equivalent residue positions in wild-type kinase structures (Spearman rank order correlation, r = 0.69, p value < 1e 139). This implies that the residues with low flexibility, like catalytic loop, do not adopt diverse fluctuations in different functional states or across kinases. Substitution with any other fluctuation is heavily disfavoured at the lower range of flexibility than at the higher range. While we did not detect apparent differences in the FLOSUMs of wild-type, disease mutant and mutants of unknown effect structures, there is a remarkable distinction in the FLOSUM of pseudo-kinases. Fluctuation substitutions that are traditionally unfavourable in wild-type kinases are freely allowed in pseudo-kinases, thus exhibit-in poor conservative substitution. Over and above the lack of conventional amino acids, poor conservation of structural fluctuations and favourable substitution of de-viand fluctuations could render auxiliary functional character to the kinase domain in pseudo-kinases, despite their structural similarity to STY kinases. Taken together, this study summarises the structural fluctuation landscape of STY kinases in the form of a substitution matrix, which can serve as a model of flexibility substitution during protein evolution. Encouraged by structural fluctuations being differentially conserved in closely re-lasted kinases (Chapter 5) and conservatively substituted across kinases (Chapter 6), we extended this principle to the sequences of STY kinases in Chapter 7. This chapter reports the development of a method to predict the sites of functional specialisation in kinases, which differentiate one kinase from another, and applies it to all known STY kinase families. These are correlates of kinase-specific functional and regulatory attributes like specific protein-protein interactions, cognate substrate recognition and response to specific signals. Two cardinal properties of family-specific functional sites, viz., differential conservation and discriminatory ability, were used to identify them. We systematically compiled a data set of 5488 kinase catalytic domain sequences be-longing to 107 families. After aligning them into a single multiple sequence alignment, we comparatively analysed the amino acid distributions in topologically equivalent positions of different families. Based on 3 different analytical measures, physicochemical property, Shannon’s entropy and random probability, we scored the differential conservation of every alignment position in each family. By maximising the disc rim-inability between the kinase families, we integrated the results of the three measures and devised a single unified scoring scheme called ID score. This integrated scoring method could distinguish the 107 families from one another with an accuracy of 99.2%. Each site in every STY kinase family was given a score in the range 0 to 1, with 0 indicating no functional specialisation and 1 indicating maximum functional spa-canalisation, by the ID score. Several validations of the method were carried out to assess its competence. First, we selected those residue positions which have consistently high ID scores across most families. Using these hotspot alignment positions that render specificity to the kinase, we clustered the kinase sequences into groups and families. We found that the ID score predicted sites clustered the kinases better than the traditional clustering using the entire alignment. Despite reduction in information, the increase in accuracy of clustering is feasible because of efficient filtering of non-discriminatory sites by ID score. Second, a linear discriminant classifier was observed to predict the kinase family, based on the ID score predicted sites, better than traditional methods. Third, family-specific protein-protein interaction sites in CDK and substrate recognising distal sites in MAPK were scored significantly higher than other residues by ID score (Two-tailed unpaired t-test, p value < 0.05). Fourth, family-specific oncogenic driver mutation sites in 8 different kinase families were identified confidently by the ID score. Finally, we demonstrate one feasible application of the ID score method in the prediction of specific protein-protein interaction sites. In summary, we developed an integrated discriminatory method to identify regions of functional specialisation in all known kinases, validated the results for known cases and elucidate a potential application of the method. The learning from the entire thesis work is summarised in Chapter 8, which positions the work in the larger context of functioning of the kinase domain and the use of dynamics to interpret protein functions. The validity of the simple, yet use-full, NMA of proteins and complementary MD simulations to understand basic mechanistic and dynamic properties of proteins is highlighted. Similar to sequence and structure, dynamics is now recognised as a crucial feature holding information about protein function. The main learning of the thesis that the flexibility and mobility of STY kinases is conserved and conservatively substituted at different levels of hierarchy (different functional forms within a kinase, across kinase families and across the entire STY kinase superfamily) is discussed. The contributions of the work in fur-the ring the knowledge of specificity determinants in kinases, which dictate precise regulatory and control mechanisms, are presented. Supplementary information helpful in understanding of the results of individual chapters, but could not be printed in the thesis due to its length, are provided in an optical disk attached to the thesis. The material in the optical disk is referred to in appropriate places in the individual chapters

Über die Proteinphosphorylierung in den Mitochondrien der Hefe Saccharomyces cerevisiae ist im Gegensatz zu anderen Kompartimenten nur wenig bekannt. Insbesondere hinsichtlich der physiologischen Bedeutung sowie den an der Modifikation beteiligten Enzymen sind kaum Daten verfügbar. Vor diesem Hintergrund stand die Identifizierung und molekularbiologische Charakterisierung mitochondrialer Proteinkinasen (PKasen) und Proteinphosphatasen (PPasen) im Fokus dieser Arbeit. Unter Verwendung komparativer 2D DIGE-Analysen konnten zwei Strategien erfolgreich verfolgt werden: Zum einen wurde die Konsequenz einer Gendeletion von ausgewählten PKasen bzw. PPasen mit putativer mitochondrialer Lokalisation untersucht. Dabei gelang es, die an der in vivo Regulation des Pyruvatdehydrogenase(PDH)-Komplexes beteiligten Enzyme erstmalig zu identifizieren und im Folgenden deren regulatorisches Zusammenspiel umfassend zu analysieren. Zum anderen wurde in einem inversen Ansatz beispielhaft für die PKase Sat4p untersucht, welche Auswirkungen eine Überexpression dieser Kinase auf das mt Proteom hat. Erste Hinweise, welche zur Identifizierung der PDH-Kinasen (Pkp1p und Pkp2p) bzw. PDH Phosphatasen (Ppp1p und Ppp2p) führten, lieferten die signifikanten Spotänderungen von Pda1p (E1α-Untereinheit des PDH-Komplexes) in den 2D-DIGE-Analysen. Im Folgenden wurde die mitochondriale Lokalisation der vier regulatorischen Enzyme unter Verwendung Epitop-getaggter Varianten nachgewiesen sowie Pda1p in unabhängigen phosphospezifischen Analysen als Target der Phosphorylierung verifiziert. Die Phosphorylierungsstelle von Pda1p konnte massenspektrometrisch dem Serin313 zugeordnet werden. PDH-Aktivitätsmessungen zeigten, dass die Phosphorylierung von Pda1p den PDH Komplex inaktiviert, während eine Dephosphorylierung zur Aktivierung führt. Dabei war der Einfluss der Deletion der PDH Kinasen bzw. der PDH-Phosphatasen unterschiedlich stark ausgeprägt. Während Ppp1p und Ppp2p partiell redundante Funktionen besitzen, lassen die Analysen komplementäre Aktivitäten von Pkp1p und Pkp2p vermuten. Eine physikalische Interaktion der beiden Kinasen wurde in vivo nachgewiesen und deutet auf die Bildung funktioneller Heteromere hin. Durch Analysen in der 2D-BN/SDS-PAGE konnte eine Assoziation der PDH-Kinasen sowie PDH-Phosphatasen mit dem hochmolekularen, etwa 8 MDa großen PDH-Komplex sowie mit PDH-Subkomplexen geringeren Molekulargewichts gezeigt werden. Die Erkenntnisse dieser Arbeit ermöglichten in Verbindung mit denen eigener Vorarbeiten die Erstellung eines Modells zur PDH-Regulation in Saccharomyces cerevisiae. Neben der Aktivitätsregulation durch die von Pkp1p/Pkp2p bzw. Ppp1p/Ppp2p katalysierte Phosphorylierung wird eine Funktion der regulatorischen Enzyme – insbesondere der PDH-Kinasen – an der Assemblierung bzw. Stabilisierung des PDH-Komplexes postuliert. Es konnte somit gezeigt werden, dass in der Hefe ein ähnlicher, aber nicht identischer Regulationsmechanismus vorliegt wie in höheren Eukaryoten. Die zweite Strategie, welche in dieser Arbeit exemplarisch für eine PKase verfolgt wurde, führte zur Identifikation einer bislang unbekannten Funktion der Kinase Sat4p in den Mitochondrien. Es konnte gezeigt werden, dass Sat4p dual lokalisiert in der cytoplasmatischen sowie mitochondrialen Fraktion vorliegt und selbst Target der Phosphorylierung ist. Die Überexpression von Sat4p führte nicht nur zu einem verminderten Wachstum auf nicht fermentierbaren Medien, sondern auch zur Beeinflussung spezifischer mitochondrialer Proteingruppen. Während die Spots der Proteine Pil1p und Lsp1p eine höhere Intensität zeigten, wiesen die Fe/S-Proteine Aco1p und Lys4p eine verminderte „steady-state“-Konzentration auf. Darüber hinaus lagen die Proteine, welche Liponsäure als prosthetische Gruppe tragen (Lat1p, Kgd2p und Gcv3p), im Tet-Sat4-Stamm vorwiegend in ihrer nicht-lipoylierten Form vor. Die Lipoylierungsstellen aller drei Proteine konnten im Wildtyp unter Nutzung von nanoLC-MS/MS erstmals experimentell bestimmt und Lys75 (Lat1p), Lys114 (Kgd2p) bzw. Lys102 (Gcv3p) zugeordnet werden. Die fehlende Lipoylierung der Proteine bzw. die verminderte Proteinkonzentration der Aconitase führte zu einer stark verminderten Aktivität der betroffenen Enzymkomplexe. Neben den in der Literatur beschriebenen putativen Funktionen von Sat4p bei der Regulation cytoplasmatischer Proteine wurde basierend auf den Erkenntnissen der Analysen eine spezifische Funktion der Kinase in den Mitochondrien postuliert. Das Modell schlägt eine Rolle von Sat4p in den späten Schritten der Maturation einer spezifischen Gruppe von mitochondrialer Fe/S-Proteinen vor. Die Beeinträchtigung der Lipoatsynthase Lip5p, welche neben Aco1p und Lys4p ebenfalls zu dieser Gruppe gehört, führt vermutlich sekundär zum beobachteten Verlust der Lipoylierung von Lat1p, Kgd2p und Gcv3p.

Calcium tolerant pig and rabbit cardiomyocytes were isolated using retrograde aortic perfusion of nominally calcium-free collagenase. Preconditioning protocols used 1 or 3 x l0-min episodes of ischemic pelleting or pre-incubation with 100 μM adenosine, followed by a 15-min post-incubation and 180-240-min ischemic pelleting. Control cells were incubated and washed in parallel with the experimental groups. Injury was assessed by determination of cell morphology, trypan blue permeability following osmotic swelling, lactate and HPLC analysis of adenine nucleotides. Preconditioned pig cardiomyocytes had a reduced rate of ischemic contracture, but protection occurred without conservation of ATP. Preconditioned rabbit cardiomyocytes were protected without significant changes in rates of ischemic contracture or ATP depletion. Incubation of ischemic cells with the protein phosphatase inhibitor, fostriecin, at PP2A-selective concentrations (0.1-10 μM), mimicked preconditioning in both rabbit and pig cardiomyocytes. In rabbits, the K(ATP) channel blocker, 5-hydroxydecanoate (5-HD), did not block preconditioning or fostriecin protection. In the pig, 5-HD blocked both preconditioning and fostriecin protection, with return of the rates of ischemic contracture to control. However, 5-HD was an effective blocker of protection only in early ischemia. Fostriecin mimicked preconditioning in the rabbit and the early responses of the preconditioned pig. Preconditioning appears associated with protein phosphorylation in both the rabbit and the pig, but major pathways leading to protection may differ in the two species.

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