Gotowa bibliografia na temat „Tyr (STY) protein kinases”

ABSTRACT SR proteins constitute a family of splicing factors that play key roles in both constitutive and regulated splicing in metazoan organisms. The proteins are extensively phosphorylated, and kinases capable of phosphorylating them have been identified. However, little is known about how these kinases function, for example, whether they target specific SR proteins or whether the kinases themselves are regulated. Here we describe properties of one such kinase, Clk/Sty, the founding member of the Clk/Sty family of dual-specificity kinases. Clk/Sty is autophosphorylated on both Ser/Thr and Thr residues, and using both direct kinase assays and SR protein-dependent splicing assays, we have analyzed the effects of each type of modification. We find not only that the pattern of phosphorylation on a specific SR protein substrate, ASF/SF2, is modulated by autophosphorylation but also that the ability of Clk/Sty to recognize different SR proteins is influenced by the extent and nature of autophosphorylation. Strikingly, phosphorylation of ASF/SF2 is sensitive to changes in Tyr, but not Ser/Thr, autophosphorylation while that of SC35 displays the opposite pattern. In contrast, phosphorylation of a third SR protein, SRp40, is unaffected by autophosphorylation. We also present biochemical data indicating that as expected for a factor directly involved in splicing control (but in contrast to recent reports), Clk/Sty is found in the nucleus of several different cell types.

Starch provides plants with carbon and energy during stressful periods; however, relatively few regulators of starch metabolism under stress-induced carbon starvation have been discovered. We studied a protein kinase Ser/Thr/Tyr (STY) 46, identified by gene co-expression network analysis as a potential regulator of the starch starvation response in Arabidopsis thaliana. We showed that STY46 was induced by (1) abscisic acid and prolonged darkness, (2) by abiotic stressors, including salinity and osmotic stress, and (3) by conditions associated with carbon starvation. Characterization of STY46 T-DNA knockout mutants indicated that there was functional redundancy among the STY gene family, as these genotypes did not show strong phenotypes. However, Arabidopsis with high levels of STY46 transcripts (OE-25) grew faster at the early seedling stage, had higher photosynthetic rates, and more carbon was stored as protein in the seeds under control conditions. Further, OE-25 source leaf accumulated more sugars under 100 mM NaCl stress, and salinity also accelerated root growth, which is consistent with an adaptive response. Salt-stressed OE-25 partitioned 14C towards sugars and amino acids, and away from starch and protein in source leaves. Together, these findings suggested that STY46 may be part of the salinity stress response pathway that utilizes starch during early plant growth.

Serine/arginine-rich (SR) proteins play essential roles in the constitutive and regulated splicing of precursor mRNAs. Phosphorylation of the arginine/serine dipeptide-rich (RS) domain by SR protein kinases such as Cdc2-like kinases (Clk/Sty) modulates their subcellular localization and activation. However, it remains unclear how these kinases and their target SR proteins are regulated by extracellular signals. Regulation of protein kinase C βII (PKCβII) pre-mRNA alternative splicing via exon inclusion by Akt2, a central kinase in insulin action, involves phosphorylation of SR proteins. Here we showed that Akt2, in response to insulin, resulted in phosphorylation of Clk/Sty, which then altered SR protein phosphorylation in concert with Akt2. Insulin-stimulated PKCβII pre-mRNA splicing was blocked by Clk/Sty and phosphatidylinositol-3-kinase inhibitors, and diabetic Akt2-null mouse tissues had impaired phospho-Clk/Sty, SR protein phosphorylation, and PKCβII expression. Furthermore, we observed that Akt2 phosphorylated several SR proteins distinct from Clk/Sty in response to insulin. Akt2-catalyzed phosphorylation of Clk/Sty and SR proteins revealed a role for both kinases in splicing regulation indicating dual functions for Akt2 in response to insulin in this pathway.

We have cloned a novel kinase (STY) from an embryonal carcinoma cell line. Sequence analysis of the STY cDNA reveals that it shares sequence homology with serine/threonine-type kinases and yet the bacterial expression product of the STY cDNA appears to have serine-, threonine-, and tyrosine-phosphorylating activities. The predicted STY protein is highly basic and contains a putative nuclear localization signal. During differentiation, two new mRNAs were detected in addition to the embryonic transcript.

We have cloned a novel kinase (STY) from an embryonal carcinoma cell line. Sequence analysis of the STY cDNA reveals that it shares sequence homology with serine/threonine-type kinases and yet the bacterial expression product of the STY cDNA appears to have serine-, threonine-, and tyrosine-phosphorylating activities. The predicted STY protein is highly basic and contains a putative nuclear localization signal. During differentiation, two new mRNAs were detected in addition to the embryonic transcript.

ABSTRACT The splicing of mammalian mRNA precursors requires both protein phosphorylation and dephosphorylation, likely involving modification of members of the SR protein family of splicing factors. Several kinases have been identified that can phosphorylate SR proteins in vitro, and transfection assays have provided evidence that at least one of these, Clk/Sty, can modulate splicing in vivo. But evidence that a specific kinase can directly affect the splicing activity of SR proteins has been lacking. Here, by using purified recombinant Clk/Sty, a catalytically inactive mutant, and individual SR proteins, we show that Clk/Sty directly affects the activity of SR proteins, but not other essential splicing factors, in reconstituted splicing assays. We also provide evidence that both hyper- and hypophosphorylation inhibit SR protein splicing activity, repressing constitutive splicing and switching alternative splice site selection. These findings indicate that Clk/Sty directly and specifically influences the activity of SR protein splicing factors and, importantly, show that both under- and overphosphorylation of SR proteins can modulate splicing.

Protein phosphorylation is the most frequent post-translational modification (PTM) that plays important regulatory roles in a wide range of biological processes. Phosphorylation mainly occurs on serine (Ser), threonine (Thr), and tyrosine (Tyr) residues, with the phosphorylated Tyr sites accounting for ~1–2% of all phosphorylated residues. Tyr phosphorylation was initially believed to be less common in plants compared to animals; however, recent investigation indicates otherwise. Although they lack typical protein Tyr kinases, plants possess many dual-specificity protein kinases that were implicated in diverse cellular processes by phosphorylating Ser, Thr, and Tyr residues. Analyses of sequenced plant genomes also identified protein Tyr phosphatases and dual-specificity protein phosphatases. Recent studies have revealed important regulatory roles of Tyr phosphorylation in many different aspects of plant growth and development and plant interactions with the environment. This short review summarizes studies that implicated the Tyr phosphorylation in biosynthesis and signaling of plant hormones.

Phosphorylated serine- and arginine-rich (SR) proteins are components of the spliceosomal complex, and have been implicated in the control of alternative splicing. Kinases that regulate the phosphorylation and possibly the intranuclear distribution of SR proteins may therefore contribute to changes in choice of splice site. We have cloned three mouse cDNAs with high sequence identity to the family of LAMMER kinases (i.e. kinases carrying the conserved signature EHLAMMERILG in the catalytic domain). A comparison of their amino acid sequences revealed two related subfamilies with high evolutionary conservation. We have compared the expression patterns of these proteins in mouse tissues and transformed cell lines with that of a previously cloned family member (mCLK1/STY), and detected various transcripts for each gene. This underlines previous findings of alternative splicing of mclk1/STY. Our results suggest that the proportions of products for each gene are regulated independently. We further demonstrate that all variants encode autophosphorylating proteins that can phosphorylate several biochemically purified SR proteins in vitro, leading to hyperphosphorylation of at least one SR protein in vivo. The observed tissue distributions and substrate specificities suggest that these kinases may all be constituents of a network of regulatory mechanisms that enable SR proteins to control RNA splicing.

The key regulator of entry into mitosis is the serine/threonine kinase p34cdc2. This kinase is regulated both by association with cyclins and by phosphorylation at several sites. Phosphorylation at Tyr 15 and Thr 14 are believed to inhibit the kinase activity of cdc2. In Schizosaccharomyces pombe, the wee1 (and possibly mik1) protein kinase catalyzes phosphorylation of Tyr 15. It is not clear whether these or other, as yet unidentified, protein kinases phosphorylate Thr 14. In this report we show, using extracts of Xenopus eggs, that the Thr 14-directed kinase is tightly membrane associated. Specifically, we have shown that a purified membrane fraction, in the absence of cytoplasm, can promote phosphorylation of cdc2 on both Thr 14 and Tyr 15. In contrast, the cytoplasm can phosphorylate cdc2 only on Tyr 15, suggesting the existence of at least two distinctly localized subpopulations of cdc2 Tyr 15-directed kinases. The membrane-associated Tyr 15 and Thr 14 kinase activities behaved similarly during salt or detergent extraction and were similarly regulated during the cell cycle and by the checkpoint machinery that delays mitosis while DNA is being replicated. This suggests the possibility that a dual-specificity membrane-associated protein kinase may catalyze phosphorylation of both Tyr 15 and Thr 14.

GTPase-activating protein (GAP) stimulates the ability of p21ras to hydrolyze GTP to GDP. Since GAP is phosphorylated by a variety of activated or oncogenic protein-tyrosine kinases, it may couple tyrosine kinases to the Ras signaling pathway. The epidermal growth factor (EGF) receptor cytoplasmic domain phosphorylated human GAP in vitro within a single tryptic phosphopeptide. The same GAP peptide was also apparently phosphorylated on tyrosine in EGF-stimulated rat fibroblasts. Circumstantial evidence suggested that residue 460 might be the site of GAP tyrosine phosphorylation. This possibility was confirmed by phosphorylation of a synthetic peptide corresponding to the predicted tryptic peptide containing Tyr-460. Alteration of Tyr-460 to phenylalanine by site-directed mutagenesis diminished the in vitro phosphorylation of a bacterial GAP polypeptide by the EGF receptor. We conclude that Tyr-460 is a site of GAP tyrosine phosphorylation by the EGF receptor in vitro and likely in vivo. GAP Tyr-460 is located immediately C terminal to the second GAP SH2 domain, suggesting that its phosphorylation might have a role in regulating protein-protein interactions.

Thesis (Ph. D.)--University of California, San Diego, 2008.
Title from first page of PDF file (viewed January 6, 2009). Available via ProQuest Digital Dissertations. Vita. Includes bibliographical references (p. 149-160).

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

Protein phosphorylation is a key cellular regulatory mechanism. Phosphorylation can either activate or inhibit the function of a particular protein. Activation of protein kinases has been implicated in response to light, pathogen attack, growth regulators, stress and nutrient deficiency in plants. Most of the intracellular signaling pathways use protein phosphorylation to create signals and conduct them further. Identification of the physiological substrates for the protein kinase enables the understanding of how the signaling networks function and how they are disturbed under adverse conditions. Identification of the physiological substrates for the kinase is limited by the low stoichiometry of protein phosphorylation inside the cell. Although, recent advances in mass spectrometric techniques have increased the identification of phosphorylated protein in the cell, the precise connection between the kinase and identified phosphorylated protein is not established. Dual-specificity kinases that phosphorylate on serine, threonine and tyrosine residues have been identified and characterized in plants. However, the in vivo substrates for most of these kinases have not been identified. Recently a manganese-dependent dual-specificity STY protein kinase (STYK) has been identified from Arabidopsis thaliana which has been suggested to play a role in plant growth, development and in systemic acquired resistance. The identification of the physiological substrate for AtSTYK may help in understanding the signal transduction pathway the kinase in involved and how it is perturbed in different physiological condition. Therefore, the main objectives of my current study are,  To identify the physiological substrates of the AtSTY dual specificity kinase (STYK). 1) Identification of the substrates by using genetic, proteomic and biochemical approaches. 2) Biochemical characterization of the substrate phosphorylation. 3) Identifying the biochemical function of the substrate protein. 4) Assessing the significance of substrate phosphorylation.

Protein phosphorylation mediated by protein kinases is the principal mechanism by which eukaryotic cellular processes are modulated by external physiological stimuli. Phosphopeptides are essential tools for the study of this process, serving as model substrates for phosphatases, as antigens for the production of antibodies against phosphorylated proteins, and as reference compounds for determining their physical parameters. The development of methods for the production of phosphopeptides has consequently attracted considerable interest over the last few years, and these endeavours have yielded reliable procedures which have now made their synthesis routine. There are two strategies used currently for the preparation of phosphopeptides: the building block approach, in which pre-formed protected phosphoamino acids are incorporated during the course of chain assembly, and the global phosphorylation method, which involves post-synthetic phosphorylation of serine, threonine, or tyrosine side-chain hydroxyl groups on the solid support. The building block procedure is certainly the more straightforward of the two approaches and has now become, owing to the availability of suitably protected phosphoamino acids, the standard method for the routine production of phosphopeptides. For the side-chain protection of phosphotyrosine in Fmoc/tBu-based solid phase synthesis, methyl, benzyl, t-butyl, dialkylamino, and silyl groups have been employed. Of these, benzyl is most useful as it is the most convenient to introduce and is rapidly removed during the TFA-mediated acidolysis step. Only the mono-benzyl ester, Fmoc-Tyr(PO(OBzl)-OH)-OH 1, is available commercially; the dibenzyl ester offers no practical benefit as it undergoes mono-debenzylation in the course of the piperidine-mediated Fmoc deprotection reaction. Also available commercially is Fmoc-Tyr(PO3H2)-OH 2. This derivative, despite having no phosphate protection, appears to work well, particularly in the synthesis of small- to medium-sized phosphopeptides; although formation of the pyrophosphate 3 can be a problem in peptides containing adjacent Tyr(PO3H2) residues. Phosphate triesters of serine and threonine are not compatible with Fmoc/tBu chemistry as they undergo β-elimination when treated with piperidine, resulting in the formation of dehydroalanine and dehydoaminobutyric acid, respectively For this reason, it was long believed that the building block approach could not be used for preparation of peptides containing these amino acids.