Browsing by Subject "Phosphorylation"
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Item Association of Protein Phosphatase 2A with S6 Kinase Is Regulated In an MTOR-Dependent Manner(2007-08-08) Mahmood, Nadir Ahmeduddin; Mumby, MarcThe mammalian target of rapamycin (mTOR) pathway senses nutrient and growth factors to regulate protein synthesis and cell growth. Strict control of the components of this pathway is essential for cells to reach an appropriate size. Aberrant mTOR signaling has been implicated in multiple diseases such as cancer, diabetes, and heart failure. Activation of mTOR by nutrients and growth factors leads to the phosphorylation of its two substrates, S6 kinase 1 (S6K1) and 4E binding protein 1 (4EBP1) to promote translation initiation. Many reports have demonstrated the rapid dephosphorylation of S6K1 following treatment with the mTOR inhibitor, rapamycin, as well as by the depletion of amino acids. These data suggest a role for a serine/threonine protein phosphatase in mediating this dephosphorylation. This was supported by studies showing the co-immunoprecipitation of the catalytic subunit of protein phosphatase 2A (PP2A) with S6K1. PP2A is a ubiquitously expressed phosphatase that has been implicated in many intracellular signaling pathways. In this dissertation, an association between S6K1 and PP2A was investigated to elucidate the role of this phosphatase in regulating nutrient signaling and cell growth. Initial studies utilized an inhibitor of PP2A and related phosphatases to block the dephosphorylation of S6K1 under conditions that inactivate mTOR. Immunoprecipitation studies identified an interaction between the PP2A catalytic and scaffolding subunits and S6K1 that was enhanced under conditions that lead to S6K1 dephosphorylation and decreased upon stimulation of the pathway by insulin. An siRNA screen targeting PP2A regulatory subunits was used to identify specific subunits that were mediating this association. Depletion of the B56gamma and B56delta regulatory subunits reduced co-immunoprecipitation of PP2A with S6K1. However, functional studies found no effect of knockdown of these subunits on the dephosphorylation of S6K at T389 or on S6K1 kinase activity. There was no effect on cell size in cells depleted of B56gamma or B56delta , but cell proliferation was reduced in these cells. It is likely that cell proliferation was affected due to the roles of these subunits in other pathways. Additionally, knockdown of PP2A-like phosphatases did not definitively identify the phosphatase that dephosphorylates S6K1 at T389.Item Characterization of a Novel Signaling Motif in the CD3 Subunit(2010-11-02T18:12:09Z) Watts, Laura Michelle; Oers, Nicolai S.C. vanThe T cell receptor (TCR) complex consists of the ligand-binding α/β heterodimer as well as four associated signaling chains (CD3 γ, δ, ε, and ζ). Each of the CD3 subunits contains one or more copies of a signaling motif termed an immunoreceptor tyrosine-based activation motif (ITAM). Phosphorylation of tyrosine residues within the ITAMs is critical for TCR-mediated signaling events. We identified an additional signaling motif in the cytoplasmic tail of CD3 ε that we termed the basic-rich stretch (BRS). Biochemical analyses revealed that this motif uniquely interacted with the serine/threonine kinase, G protein-coupled receptor kinase 2 (GRK2). Interactions between the BRS and GRK2 contribute to the ability of the TCR to cross talk with G protein-coupled receptors, such as CXCR4. The BRS was also capable of mediating interactions with certain charged phospholipids. To address the role of the BRS in T cell functions, several murine CD3 ε transgenic lines bearing distinct mutations of the BRS were generated. Analyses of these mice on a CD3 ε null background revealed that modifications of the BRS suppressed T cell development. Taken together, these findings demonstrate that the BRS of CD3 ε plays an important role in TCR signaling and T cell development by regulating unique protein-protein and protein-lipid interactions.Item Characterization of the Protein Phosphatase 2A Regulatory Subunit PR70(2005-12-19) Davis, Anthony John; Mumby, Marc C.Protein phosphatase 2A (PP2A) is a phosphoserine/threonine phosphatase that controls the phosphorylation of numerous proteins in eukaryotic cells. PP2A consists of a core dimer composed of a scaffolding subunit (A-subunit) and a catalytic subunit (C-subunit) that interacts with a variety of regulatory subunits. There are four families of regulatory subunits: R2, R3, R4, and R5. The diversity of regulatory subunits gives rise to multiple PP2A holoenzymes and accounts for the ability of PP2A to regulate diverse cellular processes. Relatively little is known about the molecular basis for the interaction of the regulatory subunits with the core dimer and substrates. A more thorough understanding of these interactions would provide insights into how the regulatory subunits target PP2A to different cellular processes. The R3 regulatory subunit termed PR70 was identified in a yeast two hybrid screen with the DNA replication protein Cdc6 as bait. PR70 interacts with the PP2A core dimer and Cdc6 in vivo and in vitro. Biochemical approaches were used to identify regions and residues within PR70 that are important for mediating protein-protein interactions with the PP2A core dimer and Cdc6. PR70 contains two conserved calcium binding EF hand motifs and binds calcium in vitro. Calcium enhances the binding of PR70 to the A-subunit but not to Cdc6. Although calcium did not enhance the binding of PR70 to Cdc6, it did result in an increase in the amount of PP2A associated with Cdc6. Both calcium binding and enhanced interactions with the A-subunit require functional EF hand motifs. A conserved motif within the conserved R3 family domain was identified that is sufficient for the interaction of PR70 with PP2A. The C-terminal region was shown to be important for the interaction of PR70 with Cdc6, but not with the A-subunit. This result suggested that different portions of PR70 are important for mediating interactions with PP2A and Cdc6. Finally, PR70 is phosphorylated in intact cells at threonine 76 and serine 543. A functional analysis indicated that mutation of these sites does not affect the ability of PR70 to interact with PP2A, suggesting phosphorylation plays some other role in regulating PR70.Item Dynamin 2 Mutations Implicated in Charcot-Marie-Tooth Disease(2010-05-14) Tassin, Tara Charisse; Albanesi, JosephDynamins are large (100 kDa) GTPases responsible for severing the necks of nascent vesicles during clathrin- and caveolae-mediated endocytosis, and are implicated in a variety of other cellular processes, including macropinocytosis, phagocytosis, and cytoskeletal organization. Mammalian cells contain three dynamin genes, encoding dynamin 1 (expressed in neurons and neuroendocrine cells), dynamin 2 (ubiquitously expressed), and dynamin 3 (enriched in testes, but also found in pre- and post-synaptic regions of neurons). Dynamin 2 was identified as a locus for Charcot-Marie-Tooth disease (CMT) and Centronuclear Myopathy (CNM). CMT is a peripheral neuropathy affecting 1 in every 2,500 people, making it one of the most commonly inherited neurological disorders. CNM causes progressive loss of muscle tone without primary neuronal involvement. In this study, the effects of two CMT mutations were characterized in order to gain insight into the causes of the disease. The two mutations, K558E and delDEE (a deletion of residues 551-553), are both located in the Pleckstrin Homology (PH) domain (approximately residues 520-630), which mediates the binding of dynamins to phosphoinositide lipids and to βγ subunits of heterotrimeric G proteins. The overall goal of the project was to determine how the mutations influence fundamental properties of dynamin 2, including: 1. Self-assembly and concentration-dependent GTPase activation; 2. Binding to phosphatidylinositol-(4,5)- bisphosphate (hereafter termed PIP2) and stimulation of GTPase activity by PIP2; 3. Stimulus-dependent tyrosine phosphorylation; and 4. Interaction with G-βγ. In summary, I have found that Dyn 2-K558E undergoes normal self-assembly and self-activation, but that its activation by PIP2-containing vesicles is drastically reduced. Consistent with this observation, the ability of the isolated K558E PH domain to bind to PIP2-containing vesicles was also impaired. Because full-length Dyn 2-delDEE could not be expressed in Sf9 cells, I was unable to determine effects of this deletion on its self-assembly, self-activation, or activation by PIP2 vesicles. However, I took advantage of the bacterially-expressed GST-tagged PH domain to demonstrate that deletion of residues DEE does not affect binding to PIP2, whereas it strongly (6-20 fold) enhances the interaction with G-βγ. This enhanced binding may be significant in explaining the role of the delDEE mutation in CMT disease, as previous studies have shown that G-βγ inhibits the GTPase activity of dynamin 1. Although full-length Dyn 2-delDEE protein could not be obtained for in vitro analysis, I was able to express the full-length mutant in mammalian cells, allowing me to examine its ability to undergo tyrosine phosphorylation. Consistently, Dyn 2-delDEE underwent approximately 2-3 fold higher levels of tyrosine phosphorylation than either wild-type dynamin 2 or Dyn 2-K558E in Src-expressing cells stimulated by EGF or isoproterenol. Mutation of the two tyrosines (individually or in combination) previously shown to be the major Src-phosphorylated sites in dynamins significantly reduced tyrosine phosphorylation in both wild-type and mutant dynamins. Finally, I compared the effects of overexpression of wild-type dynamin 2 and Dyn 2-delDEE on stimulus-dependent activation of the MAP kinases Erk1/2. These experiments were motivated by earlier studies indicating that maximal Erk activation cannot occur if receptor-mediated endocytosis is inhibited. Overexpression of Dyn 2-delDEE reduced Erk activation by 70%, and activation was further reduced by mutation of the two phosphorylatable tyrosines. Mutation of the phosphorylatable tyrosines in wild-type dynamin 2 resulted in a 50% inhibition of Erk activation. Overall, the results of my analysis demonstrate that two CMT mutations within the same domain of dynamin 2 have distinctly different properties. Future studies will be aimed at determining if these mutants impair endocytosis by distinct mechanisms.Item Evaluation and Characterization of Novel Signal Transduction Pathways in Striatum(2008-05-13) Sahin, Bogachan; Bibb, James A.In the mammalian central nervous system, protein kinases and protein phosphatases control the function of myriad target proteins in the pre- and postsynaptic compartments, including other protein kinases and phosphatases, neurotransmitter receptors, ion channels, transporters, metabolic enzymes, transcription factors, cytoskeletal elements, and vesicle-docking proteins. Using biochemical and pharmacological approaches, a number of novel striatal signal transduction pathways were evaluated and characterized in the following studies, with emphasis on protein kinase C-mediated signaling. 1) A known and novel form of mouse Adk encoding splice variants of adenosine kinase, the principal enzyme of adenosine metabolism, were cloned from a mouse brain cDNA library and expressed and purified as recombinant proteins with high enzymatic activity. The tissue distribution of adenosine kinase isoform expression was defined. A polyclonal anti adenosine kinase antibody was generated for further characterization of the enzyme. In vitro protein phosphorylation studies using purified protein kinases and in vivo radioimmunoprecipitation assays using the novel antibody for adenosine kinase indicated, however, that this metabolic enzyme is unlikely to be regulated by phosphorylation. 2) Further studies using a candidate approach demonstrated the regulation of several postsynaptic phosphoproteins by striatal adenosine A2A receptor signaling, including ionotropic glutamate receptor subunits, mitogen-activated protein kinase isoforms, a striatal inhibitor of protein phosphatase 1, a protein phosphatase 1- and actin-binding protein, and the cAMP-response element-binding protein. 3) In parallel studies, inhibitor-1, a protein phosphatase 1 inhibitor activated by cAMPdependent protein kinase, was characterized as a novel protein kinase C substrate in vitro and in vivo. Phosphorylation state-specific antibodies raised against this novel phosphorylation site showed that it is dephosphorylated by protein phosphatase 1 and positively regulated by group I metabotropic glutamate receptors in the striatum. Furthermore, protein kinase C-dependent phosphorylation was shown to reduce the efficiency with which inhibitor-1 serves as a substrate for cAMP-dependent protein kinase in vitro and in vivo. 4) Finally, protein kinase C activation was shown to decrease the level of phosphorylation of cyclin-dependent kinase 5 substrates in the striatum, suggesting a possible role for protein kinase C in regulating cyclin-dependent kinase 5 activity.Item The Functional Characterization of the Quorum Sensing E. Coli Regulators B and C in EHEC(2005-12-19) Clarke, Marcie B.; Sperandio, VanessaEnterohemorrhagic E. coli (EHEC) is the causative agent of hemorrhagic colitis. During an infection, EHEC can sense and respond to environmental cues, including the cell density of the intestinal normal flora (through the floral-derived AI-3 signal) and the epinephrine/norepinephrine produced naturally by the host. This cell-to-cell signaling may aid in colonization and disease by allowing EHEC to up-regulate its flagella and motility genes to swim closer to the intestinal epithelium. Previously, Sperandio et al. (2002) have shown that the quorum sensing E. coli regulators B and C (QseB&C), a two-component system in EHEC, are responsible for the regulation of the master regulator of flagella and motility genes, flhDC, in response to cell-to-cell signaling [1]. Here, we show that QseC, the membrane-bound sensor kinase, can autophosphorylate itself in response to AI-3 or epinephrine/norepinephrine and then transfer this phosphate to the response regulator, QseB. The autophosphorylation of QseC is not affected by the addition of autoinducer-2 or intestinal hormones, including gastrin, galanin, and secretin. Additionally, autophosphorylation can be antagonized upon the addition of phentolamine, an a-adrenergic receptor antagonist. Given that enterocytes harbor a-adrenergic receptors, it would be consistent for a microbial adrenergic sensor (QseC) to mostly resemble (in an orthologous and not a homologous fashion) an a- and not a ᭡drenergic receptor. Taken together, these results suggest that QseC may be a microbial adrenergic receptor conserved amongst different bacterial and fungal species. After QseC has autophosphorylated and transferred its phosphate to QseB, QseB acts as a transcription factor to activate the expression of flhDC, the master regulator of flagella and motility genes. Nested deletion analyses of the flhDC promoter suggest that QseB may bind to three promoter regions, to either repress or activate transcription. Further transcriptional studies suggest that phosphorylated QseB autoregulates its own transcription in a similar manner. These analyses have identified a QseB consensus binding sequence, which was utilized in an in silico search to identify novel potential targets of QseB. Through the use of both biochemistry and genetics, a comprehensive model of the QseB&C signaling cascade was generated.Item Interaction Mapping of the Atypical Protein Kinase WNK3(2009-09-04) Self, Jon Tate; Cobb, Melanie H.The story of the protein kinase "with no lysine 3" (WNK3) represents a unique chapter in the larger story of protein kinases, the so-called 'molecular switches' of the cell that serve the vital function of phosphorylating myriad proteins. In doing so, these enzymes furnish the cell with one of the primary means by which signals from the external environment are transduced into cellular consequences. At the time our lab reported discovery of the first WNK, it was thought that all protein kinases contained an invariant catalytic lysine necessary for phosphoryl transfer in ß strand 3 (protein kinase subdomain II) of the highly conserved catalytic domain. Analysis of WNK1 uncovered a cysteine in the place of the so-called canonical catalytic lysine--hence the name WNK for "with no lysine". Subsequently, other WNKs came to light, and together with WNK1, they comprise an atypical branch of the kinome--the functions and significance of which are still being elucidated. Of clinical significance, WNKs 1 and 4 have been implicated in a heritable form of hypertension (pseudohypoaldosteronism type II). WNK3 has been reported to regulate certain members of the SLC12A family of cation/Cl- cotransporters (KCC1/2; NKCC1; NCC), and also to localize to various Cl- transporting epithelia and certain brain neurons with GABA-A ionotropic receptors. My goal with these interaction mapping efforts has been to build a collection of putative WNK3 interactors to serve as a source of information and project leads for the ongoing research program of the Cobb laboratory. The yeast two-hybrid screens described here have yielded hundreds of putative interactors. While this written work deals only with a small number of the most interesting putative interactors, together they point toward a number of unexpected roles for WNK3, including putative interactions with RNA-binding proteins, transcriptional regulators and proteins implicated in developmental disorders and neurodegenerative disease. The story of the WNK kinases will go on. With a connection to ion flux diseases well-established, the WNK family will surely continue to attract attention for many years, particularly given their potential as drug targets.Item ISGylation and phosphorylation : two protein posttranslational modifications that play important roles in influenza A virus replication(2008-08) Hsiang, Tien-ying, 1976-; Krug, Robert M.Two posttranslational modifications, ISGylation and phosphorylation, impact the replication of influenza A virus, a human pathogen responsible for high mortality pandemics. The ubiquitin-like ISG15 protein is induced by type 1 interferon (IFN) and is conjugated to many cellular proteins by three enzymes that are also induced by IFN. Experiments using ISG15-knockout (ISG15-/-) mice established that ISG15 and/or its conjugation inhibits the replication of influenza A virus, but inhibition was not detected in mouse embryo fibroblasts in tissue culture. The present study is focused on the effect of ISG15 and/or its conjugation on the replication of influenza A virus in human cells in tissue culture. IFN-induced antiviral activity against influenza A virus in human cells was significantly alleviated by blocking ISG15 conjugation using small interfering RNAs (siRNAs) against ISG15 conjugating enzymes. IFN-induced antiviral activity against influenza A virus gene expression and replication was reduced 10-20-fold by suppressing ISG15 conjugation. Unconjugated ISG15 does not contribute to this antiviral activity. Consequently human tissue culture cells can be used to delineate how ISG15 conjugation inhibits influenza A virus replication. SiRNA knockdowns were also used to demonstrate that other IFN-induced proteins, specifically p56, MxA and phospholipid scramblase 1, also inhibit influenza A virus gene expression in human cells. The research on phosphorylation focused on the viral NS1A protein, a multifunctional virulence factor. Although threonine phosphorylation of the NS1A protein was discovered 30 years ago, the sites of phosphorylation and its function had not been identified. A recombinant influenza A virus encoding an epitope-tagged NS1A protein was generated, enabling the purification of NS1A protein from infected cell extracts. Mass spectrometry identified phosphorylation at T49 and T215. A recombinant virus in which phosphorylation at 215 was abolished by replacing T with A is attenuated, and an apparently aberrant NS1A protein is produced. Attenuation did not occur when T was changed to E to mimic a constitutively phosphorylated state, or surprisingly when T was changed to P to mimic avian NS1A proteins. These results suggest that T215 phosphorylation in human viruses and P215 in avian viruses can support analogous functions.Item Mechanism and Regulation of ERK2 Subcellular Localization(2004-05-04) Whitehurst, Angelique Wright; Cobb, Melanie H.Dynamic changes in the localization of activated proteins can be obligatory events in signaling networks that control cell behavior. ERK1/2 activation contributes to regulated processes such as proliferation, differentiation and survival through the phosphorylation of multiple nuclear and cytoplasmic substrates. The pleiotropic effects of ERK1/2 activation suggest that regulated compartmentalization of the kinases and substrates may contribute to the fidelity of phenotypic changes in response to specific cell stimuli. Therefore, elucidating the mechanism of translocation as well as how this process is controlled is important for understanding how MAP kinases transmit signals. In vitro studies using a permeabilized cell system indicate that nuclear import of ERK2 is not regulated by soluble transport factors, but requires access to nucleoporins. While this process is not influenced by classical import machinery, it can be modulated by anchoring proteins that bind to ERK2 and sequester the kinase in the cytoplasm. One of these proteins, PEA-15, prevents ERK2 import in an in vitro system by inhibiting the kinases' ability to interact with nucleoporins. In vivo assays of phosphorylated ERK1/2 show discrete subcellular localization patterns in response to different stimuli that are independent of the level of ERK1/2 activation. Under conditions in which ERK1/2 is concentrated in the cytoplasm, the nuclear substrate of the kinase, c-Fos, is not expressed, while the cytoplasmic substrate of ERK1/2, p90RSK, is phosphorylated.Item Phosphorylation of hexokinase by tyrosine and serine/threonine specific kinases(Texas Tech University, 1992-12) Seale, Jeffrey WadeNot availableItem Rhodopsin kinase structure: different nucleotide-binding states and implications for mechanism of activation of a G protein coupled receptor kinase(2007-12) Singh, Puja, 1979-; Tesmer, John; Hackert, Marvin L.G protein coupled receptor (GPCR) kinases (GRKs) phosphorylate activated heptahelical receptors, leading to their uncoupling from G proteins and downregulation. The desensitization of GPCRs is critical to render cells responsive to further stimuli and if not regulated can result in many pathophysiological processes including heart abnormalities and hypertension. How GRKs recognize and are activated by GPCRs are not known, in part because the critical N-terminus and the kinase C-terminal extension were not resolved in GRK2 and GRK6 structures. The long-term goal of this project was to address this question by structural analysis of rhodopsin kinase (also known as GRK1), which represents a model system for studying phosphorylation-dependent desensitization of activated GPCRs. Herein we report structures of GRK1 from six crystal forms that represent three distinct nucleotide-ligand binding states. One of the (Mg²⁺)₂·ADP·GRK1 structures is the most high-resolution structure (1.85 Å) of a GRK to date. In one (Mg²⁺)₂·ATP·GRK1 structure, almost the entire N-terminal region (residues 5-30) is observed. In addition, different segments of the kinase C-terminal extension are ordered in the various nucleotide-bound structures. Together, these two elements form a putative receptor-docking site adjacent to the hinge of the kinase domain. Based on these structures, a model is proposed for how GRK1 interacts with activated rhodopsin and how rhodopsin binding in turn could activate the kinase. Two novel phosphorylation sites were also identified at the N-terminus. The physiological role of phosphorylation sites and the extensive dimerization interface mediated by the regulator of G protein signaling (RGS) homology domain of GRK1 was assessed using site-directed mutagenesis. In addition to mediating interaction with activated GPCRs, the N-terminus of GRKs also forms a binding site for calcium sensing proteins. Although its physiological significance is debated, the structures of these complexes could lend further insights into the conformation of the N-terminus of GRKs. The second chapter deals with attempts to isolate Ca²⁺·recoverin·GRK1 and Ca²⁺·calmodulin·GRK6 complexes. Finally, the RH domain of GRK2 binds G[alpha subscript q], G[alpha]₁₁, and G[alpha]₁₄ subunits thereby blocking their interactions with the downstream effectors. The third chapter involves attempts to isolate a complex of GRK6 and G[alpha]₁₆, a member of G[alpha subscript q] family.Item Signal Specific Ubiquitination and Degradation of IkBa(2003-10-08) Hakala, Kevin William; Kodadek, ThomasThe transcription factor Nuclear Factor kB (NF-kB) is retained in the cytoplasm by the action of its inhibitor IkB. Upon phosphorylation by the IKK complex, IkB is rapidly ubiquitinated and targeted for 26S proteasome mediated degradation, thus liberating NF-kB for transport to its nuclear destination. The current project was initiated to reconstitute this pathway in vitro by using the purified ubiquitination and degradation machinery to degrade IkBa, and activate NF-kB. While signal dependant IkBa ubiquitination was achieved early in the project, this substrate was not degraded by a number of different 26S protein preparations. Instead, an integral or associated isopeptidase activity was observed with each 26S preparation. The development of new 26S protein purification methods has enabled the isolation of highly purified 26S proteins that exhibits low degradative activity towards the ubiquitinated IkBa substrate without excess isopeptidase activity. In an effort to increase substrate degradation, the IkBa ubiquitination reaction was carefully scrutinized. The current literature reports that Ubch5 is the relevant E2 that works in conjunction with the IkBa SCFᔲCP E3 complex, however, Cdc34/Ubc3 can also ubiquitinate IkBa, and may also be a relevant E2. While both E2s carry out in vitro signal dependant ubiquitination of IkBa, the ubiquitin conjugates made by Ubc3 are specific for Lysine-48 linked isopeptide bonds, whereas Ubch5 is able to utilize a variety of ubiquitin surface Lysine residues in isopeptide bond formation. Because K-48 linked ubiquitin conjugates are believed to target substrates for 26S mediated degradation, it was not surprising to find that my 26S proteasome preparations exhibited higher levels of IkBa degradation when ubiquitin conjugation reactions were carried out with Ubc3 instead of Ubch5. Using small interfering RNA to knock down the protein levels of each E2 in vivo, we have found that Ubc3 has no effect on IkBa degradation, whereas the Ubc5/7 double knockdown exhibits partial inhibition of IkBa degradation which is comparable to knocking down the levels of the IkBa E3 specificity factor ᔲCP. The completion of this project has established an in vitro ubiquitination and degradation system that will be instrumental for future studies aimed at determining how the 26S proteasome unfolds and degrades its protein substrates.Item Studies Of Smoothened In Hedgehog Signaling Pathway(2006-12-20) Tong, Chao; Jiang, JinThe Hedgehog (Hh) family of morphogens controls cell growth and patterning in both vertebrates and invertebrates. Malfunction of Hh signaling has been implicated in numerous human disorders. As the Hh signal transducer, the seven-transmembrane protein Smoothened (Smo) is highly regulated. It is still a mystery how Smo transduces graded Hh signal to downstream components. Although Smo shares some structural similarity with G protein coupled receptors (GPCR), there is little evidence that G proteins are involved in Hh signal transduction in physiological settings. A kinesin like protein Costal2 (Cos2) and a serine/threonine kinase Fused (Fu) form complexes with the transcription factor Cubitus-interrupts (Ci), which is essential for Hh signal transduction. However, how Smo transduces Hh signal to this complex is still not clear. In this study, we found that Smo interacts with Cos2-Fu complex through its C-terminal tail, which is essential for the Hh pathway activation. In response to Hh, Smo is phosphorylated and accumulated on the cell surface. However, the kinases responsible for Hh induced Smo phosphorylation are still unknown. It is also not clear whether phosphorylation regulates Smo activity or not. In this study, I found that protein kinase A (PKA) and casein kinase I (CKI) regulate Smo cell surface accumulation and activity in response to Hh. PKA and CKI phosphorylate Smo directly at multiple sites which form three clusters in Smo C-terminal tail. In cooperation with Jianhang, we found that phosphorylation deficient forms of Smo failed to accumulate on the cell surface and were unable to transduce Hh signal. By contrast, phosphorylation mimicking forms of Smo have increased cell surface accumulation and constitutive activity. In addition, we also found the levels of Smo cell surface accumulation and activity correlate with its phosphorylation levels, suggesting that the graded Smo activity may be regulated by differential phosphorylation of its C-terminal tail. Furthermore, I have identified multiple Arginine clusters in Smo the C-terminal tail that negatively regulate Smo activity by preventing Smo cell surface accumulation and keeping Smo C-terminal tail in a closed inactive conformation maintained by intramolecular electrostatic interactions. I have also found that the number of arginine clusters is reversely correlated with Smo cell-surface expression and activity. I also provided evidence that phosphorylation antagonizes the negative effects of the Arginines by neutralizing the positive charges they carry, which lets Smo C-terminal tail adopts an open and active conformation and promotes Smo cell surface accumulation. Based on these data, we proposed that multiple arginine clusters provide a way to finetune Smo activity in response to different Hh levels by differentially phosphorylating Smo C-terminal tail. This study also showed that Gprk2, a G protein coupled receptor kinase (GRK), plays a positive role in regulating Hh signalling. I provided evidence that Gprk2 interacts with Smo Ctail. Furthermore, I identified a new CKI phosphorylation cluster that appears to be critical for Smo endocytosis and activation.Item Understanding Postranslational Modifications Involved in Adi3 Programmed Cell Death Signaling(2012-10-15) Avila Pacheco, Julian Ricardo, 1983-Programmed cell death (PCD) is an active process by which organisms coordinate the controlled destruction of cells. In tomato, the protein kinase Adi3 (AvrPto-dependent Pto-interacting kinase 3), acts as a negative regulator of PCD and shares important functional homologies with the mammalian anti-apoptotic AGC kinase PBK/Akt. Adi3 was originally identified as an interactor of the complex formed by the tomato resistance protein Pto and the Pseudomonas syringae pv. tomato (Pst) effector protein AvrPto. The complex formed by AvrPto and Pto causes a resistance response characterized by a rapid form of PCD that limits the spread of Pst and prevents the onset of the tomato bacterial speck disease. In an effort to characterize the mechanisms by which Adi3 regulates PCD, we identified Adi3 interacting partners in a Y2H screen. Here, I describe the interaction of Adi3 with two interacting partners identified: the Sucrose Non-fermenting (SNF1) kinase complex (SnRK) which is a eukaryotic master regulator of energy homeostasis and the E3 RING Ubiquitin ligase AdBiL. Using a combination of in vitro and in vivo approaches I found that AdBiL is an active ubiquitin ligase that ubiquitinates Adi3. Interestingly, Adi3 was found to be degraded in a proteasome-dependent manner suggesting ubiquitination could play a role in its degradation. On the other hand, Adi3 was found to inhibit the SnRK complex by directly interacting with its catalytic subunit as well as by phosphorylating the regulatory subunit SlGal83 at Ser26. SlGal83 is phosphorylated at multiple sites in vivo, and this phosphorylation state, as well as its intracellular localization was found to depend on a myristoylation signal present at its N-terminus. Phosphorylation at Ser26 by Adi3 was found to alter the localization of this subunit in a myristoylation-dependent manner. The interactions studied in this dissertation provide additional evidence on the functional homologies shared by Adi3 and PKB. In addition, the regulatory control of SnRK activity and cellular localization offers a novel connection between pathways involved in energy homeostasis and pathogen-mediated PCD.