Browsing by Subject "Ubiquitin"
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Item Calpain 9 functions in TNF receptor mediated apoptosis(2012-07-20) Kunkel, Gregory Thomas; Wang, XiadongEvasion of apoptosis is a hallmark of cancer development. The Inhibitor of Apoptosis Proteins, IAPs, block Caspase activity and cell death. Release of the Second Mitochondria-Derived Activator of Caspases, Smac, from the mitochondria relieves IAP Caspase inhibition, activating apoptosis. Our lab has developed a small molecule Smac mimetic. Surprisingly, approximately 25% of cell lines show single agent Smac mimetic sensitivity through activation of autocrine TNF-a secretion and TNF dependent apoptosis. Using Smac mimetic sensitivity as a model system, I performed a genome-wide high-throughput siRNA screen and identified Calpain 9, CAPN9, as a novel component of TNF-alpha induced apoptosis. CAPN9 knockdown does not affect TNF-a secretion, yet is essential for downstream activation. Two splice variants are reported for CAPN9. The smaller splice, CAPN9-SP2, is required for effective TNF-a induced apoptosis. CAPN9 is essential for RIPK1 recruitment and ubiquitination at the TNFR1 upon activation with TNF-alpha. CAPN9 knockdown demonstrates previously unreported association of ubiquitinated proteins, and actin binding proteins with TNFR1 in the absence of stimulus. This interaction is CAPN9 dependent and correlates with CAPN9 regulation of TNF-a induced apoptosis. [Keywords: Calpain, SMAC, IAR, TNF, ubiquitin, RJPK1]Item Cbl-b: its role of expression and regulation in T-lymphocyte activation and ageing(2007) Xu, Zhun, 1973-; Jolly, Christopher A.The aging process is strongly associated with decreased activity in the immune system. Dysregulation of T-lymphocyte function, such as reduced proliferation, is one problem faced by most elder people, which prevents them from successfully dealing with exogenous pathogens. Effective regulation of T-lymphocyte activity depends on the proper and prompt transduction of both positive and negative signals within Tlymphocytes and reflects the balance between positive and negative effects. Decline of positive signaling in aging has been studied and reported, while mechanisms concerning up-regulation of negative signaling with age and its role in immune senescence are still unclear. Cbl-b, an E3 ubiquitin ligase, was studied by our lab since it regulates the ubiquitin process, a protein modification process that has suppressive effects on signaling pathways. We first determined the reaction of Cbl-b to different stimuli in young rat splenic T-lymphocytes, and showed that there is a decrease in Cbl-b protein expression upon CD28 stimulation and such protein degradation is proteasome-dependent only. We also showed the mechanism of Cbl-b expression regulation involves the intracellular movement of Nedd4 toward Cbl-b and an up-regulation of Nedd4 expression. Then we proved that in old splenic T-lymphocytes, decreased proteasome activity was unable to down-regulate the Cbl-b protein. High levels of Cbl-b in old T-lymphocytes are functional in preventing PI3K activity and are associated with reduced T-lymphocyte proliferation upon regular stimulation. T-lymphocytes from old Cbl-b knock-out mice show similar proliferative reaction to CD3 stimulation as T-lymphocytes from young wild-type, which establishes the causeeffect relationship between sustained Cbl-b expression and decreased T-lymphocyte proliferation. In summary, these data suggest a unique role of Cbl-b in regulating Tlymphocyte signal transduction and provide critical preliminary data for extending Cbl-b studies into other fields, such as carcinogenesis.Item Characterization of Herc5: the major ligase for ISG15, an antiviral ubiquitin-like protein(2007-08) Dastur, Anahita R., 1975-; Huibregtse, Jon M.Human ISG15 is a 17 kDa ubiquitin-like protein (Ubl) that is induced by type I interferons (interferons [alpha] and [beta]) and plays a role in antiviral responses. ISG15 is conjugated via its C-terminus to more than 150 cellular proteins, and like ubiquitin, an E1-E2-E3 enzymatic cascade is required for conjugation. Ube1L and UbcH8 were previously identified as the E1 and E2 enzymes for this pathway. My experiments identified Herc5, a HECT domain E3, as the major ligase for ISG15. Like ISG15, Ube1L, and UbcH8, expression of Herc5 is transcriptionally induced by type I interferons. siRNAs against Herc5 abrogated ISG15 conjugation to the vast majority of target proteins in interferon-treated cells. Wild type Herc5, but not the catalytically inactive C994A mutant, supported conjugation of ISG15 in non-interferon-treated cells co-transfected with Ube1L, UbcH8 and ISG15. IQGAP1, a scaffold protein, was identified as another essential component of the ISG15 system. IQGAP1 was discovered to interact with Herc5, and this interaction was mediated by the C-terminal domain of IQGAP1 and the N-terminal RCC1-like repeats of Herc5. IQGAP1 was required for auto-conjugation of ISG15 to Herc5, and I propose a model where IQGAP1 functions, at least in part, by relieving an auto-inhibitory conformation of Herc5. Thus, I have identified two factors that are critical for ISG15 conjugation and my discoveries have increased our understanding of the ISG15 pathway. Identification and characterization of the conjugation apparatus will aid in establishing an in vitro biochemical system for ISG15 conjugation, which in turn, will be important to decipher the biological function of ISG15 modification.Item Characterizaton of Mcl-1 in Regulating Different Forms of Cell Death(2007-12-17) Gao, Wenhua; Wang, XiaodongProgrammed Cell Deaths (apoptosis, autophagic cell death and necrosis) play essential roles in animal development and certain diseases. Autophagy is a cellular process that provides nutrients to starved cells by digesting cellular constituents. Defects in autophagy have been implicated in cancer and neurodegeneration, but how autophagy causes cell death is not understood. In mammals, there are two apoptotic pathways: the extrinsic one through death receptors in the cell membrane and the intrinsic one through mitochondria that release death proteins. The Bcl-2 family of proteins function upstream in the intrinsic pathway. They can be subdivided into anti-apoptotic and proapoptotic proteins. Mcl-1 has two different splice variants, Mcl-1L and Mcl-1S. Mcl-1L is an anti-apoptotic Bcl-2 family protein with a short half-life, which functions in the apical step of the intrinsic apoptotic pathway and can inhibit the release of death proteins from mitochondria. After genotoxic treatment, Mcl-1L is rapidly degraded, resulting in mitochondria damage and apoptosis. Prevention of Mcl-1L degradation with proteasome inhibitors blocks apoptosis. An Mcl-1L ubiquitin ligase was identified using biochemical purification method and named Mule, which has five recognizable domains including UBA, WWE, HECT and two ARM repeats like domains. Mule also contains a region similar to the Bcl-2 homology region 3 (BH3) that allows it to specifically interact with Mcl-1L. Depletion of Mule by RNA interference stabilizes Mcl-1L, resulting in an attenuation of apoptosis induced by DNA-damaging agents. To further understand Mcl-1 function, I generated inducible Mcl-1 knockdown stable U2OS cell lines. Depletion of Mcl-1L but not Mcl-1S causes the cells to commit autophagic cell death. The autophagic phenotype can be blocked by knocking down Beclin 1, which is a known upstream component of the autophagic pathway. Mcl-1L knockdown induced cell death is accompanied by and requires the upregulation of p53 and p21. Loss-of-function experiments confirmed the involvement of Mcl-1S in inducing autophagy. Taken together, my data showed that Mcl-1 genes function in the apical step of both apoptosis and autophagic cell death pathways.Item The determinants of chain type specificity and the mechanism of polyubiquitination by HECT E3s(2010-12) Kim, Hyung Cheol; Huibregtse, Jon M.; Appling, Dean; Johnson, Arlen; Paull, Tanya; Stevens, ScottUbiquitination is a post-translational modification that can take several forms. Some proteins are modified with a single ubiquitin molecule, while others are modified with polyubiquitin chains. Each type of ubiquitination is thought to have distinct biological functions. The best-characterized types of ubiquitin modification are K48-linked polyubiquitination, which serves as a signal for proteasomal degradation and K63-linked polyubiquitination, which has non-proteolytic functions such in DNA repair, signaling, and endocytosis. HECT ubiquitin ligases (HECT E3s) form a class of E3s, defined by a C terminal catalytic domain. Several lines of evidence suggested that the HECT E3s assemble a polyubiquitin chain in a sequential manner with one molecule of ubiquitin at a time being conjugated to the distal ubiquitin of the chain. In the process of chain elongation, not all HECT E3s target a common internal lysine of ubiquitin, leading to diversification of chain type specificity in HECT E3s. For example, yeast Rsp5 forms K63 chains, while human E6AP forms K48 chains. Two important mechanistic questions were addressed in my work: 1) what are the determinants of chain type specificity of HECT E3s, and 2) what allows the distal ubiquitin of a chain to be continuously oriented near the active site of the HECT domain in the course of a sequential polyubiquitination reaction? I have determined that the chain type specificity of Rsp5 is a function solely of the HECT domain. Further, through the generation of chimeric HECT E3s, I demonstrated that chain type specificity determinants are located within the last 60 amino acids of the C lobe of the HECT domain. To address the second question, we solved the structure of Rsp5 HECT domain in complex with non-covalently bound ubiquitin in collaboration with Jue Chen’s laboratory (Purdue University). From the structure, we found that the N lobe of the HECT domain binds ubiquitin in a manner distinct from other known ubiquitin binding domains, and I have shown that Rsp5 proteins defective for ubiquitin binding are defective for chain elongation. We hypothesize that the ubiquitin binding site functions in the recruitment of the distal ubiquitin of polyubiquitin chain for efficient polyubiquitination.Item The enzymology and substrate selectivity of the ISG15 conjugation system(2010-05) Durfee, Larissa Anne; Huibregtse, Jon M.; Krug, Robert; Russell, Rick; Sawyer, Sara; Sullivan, ChrisISG15 is an interferon-induced and anti-viral ubiquitin-like protein (Ubl). Ube1L, UbcH8, and Herc5 have been identified as the E1-E2-E3 enzymes for ISG15 conjugation, and, like ISG15, their expression is induced by type I interferons. Although Herc5 is the major E3 for ISG15, over 300 proteins have been identified as ISG15 target proteins in interferon-stimulated cells. In this work, I address two aspects of the human ISG15 conjugation system: 1) the specificity of the Ube1L-UbcH8 interaction and 2), the basis of substrate recognition by Herc5. Regarding the selection of UbcH8 by Ube1L, my experiments show that although UbcH8 had been reported to function as an E2 for both Ub and ISG15, UbcH8 is preferentially activated by Ube1L compared to Ube1 (E1[superscript Ub]). The basis of this preference is a result of specific interactions between the ubiquitin-fold domain (UFD) of Ube1L and the amino-terminal [alpha]1 helix and [beta]1 [beta]2 region within UbcH8. Examination of the interferon-induced and transfected expression levels of UbcH8, combined with the kinetic constants, suggest that UbcH8 is unlikely to function as a Ub E2 in most cell lines. In examining the selection of target proteins by Herc5, I show that the range of substrates extends far beyond the proteins identified in proteomics studies and includes many exogenously expressed foreign proteins. Furthermore, I show that ISG15 conjugation is restricted to newly synthesized pools of proteins and Herc5 is associated with polyribosomes. I propose a model for ISGylation in which Herc5 broadly modifies newly synthesized proteins in a co-translational manner and suggest that, in the context of an interferon-stimulated cell, newly translated viral proteins may be primary targets of ISG15. Consistent with this, I show that ISGylation of human papillomavirus (HPV) L1 capsid protein has a dominant-inhibitory effect on the infectivity of HPV16 pseudoviruses. These discoveries have greatly increased our understanding of the mechanism of ISG15 pathway and provide a framework for establishing an in vitro ISG15 conjugation system and further examination of the anti-viral function of ISG15.Item Identification and Characterization of a New E1 that Activates Ubiquitin and FAT10(2008-05-13) Chiu, Yu-Hsin; Chen, Zhijian J.Ubiquitination is one of many post-translational modifications in eukaryotes. Three enzymes (E1, E2, and E3) are involved in conjugating ubiquitin to protein substrates. I identified a novel E1-like protein, E1-L2, which is homologous to the ubiquitin E1 and another E1 involved in the activation of the ubiquitin-like protein ISG-15 (E1-L1). E1-L2 activates both ubiquitin and FAT10, a ubiquitin-like protein. Interestingly, E1-L2 can transfer ubiquitin to only a subset of E2 enzymes, Ubc5 and Ubc13, but not Ubc3 and E2-25K, suggesting that E1-L2 may function in certain ubiquitination reactions. E1-L2, but not E1 or E1-L1, forms a thioester with FAT10 in vitro. The formation of the thioester bond requires the active site cysteine residue of E1-L2 and the C-terminal diglycine motif of FAT10. In addition, endogenous FAT10 forms a thioester with E1-L2 in cells stimulated with tumor necrosis factor-alpha and interferon-gamma, which induce FAT10 expression. Silencing of E1-L2 expression by RNAi impaired the formation of FAT10 conjugates in cells. Furthermore, E1-L2 deficient embryos died before embryonic day 13.5, suggesting that E1-L2 is essential for early embryonic development. Since the FAT10-deficient mice develop normally, it is likely that specific ubiquitination reactions catalyzed by E1-L2 play an important role in animal development.Item Mechanistic Studies of the Activation of Ubiquitin-Conjugating Enzymes by Ring-Type Ubiquitin Ligases(2006-05-15) Ozkan, Engin; Deisenhofer, JohannUbiquitination, modification with ubiquitin, is a post-translational regulation of proteins in eukaryotes. Ubiquitin-activating enzymes (E1) activate ubiquitin and form thioester linkages with ubiquitin, which are then transferred onto ubiquitin-conjugating enzymes (E2). Ubiquitin-conjugating enzymes, with the help of ubiquitin ligases (E3) transfer ubiquitin onto ubiquitination targets to form isopeptide linkages between the targets and ubiquitin. The function of ubiquitin ligases in the ubiquitination process has not been clearly understood. Recently, it has been shown that ubiquitin ligases are modular proteins or protein complexes that bring together the ubiquitination targets and the E2 enzyme. In this study, we show that E3 enzymes without their target-binding domains are effective activators of the ubiquitination reaction. We demonstrate that E3 binding to E2 enzymes increases the rate of the breakdown of the E2-ubiquitin thioester bond. Using the Statistical Coupling Analysis, a pairwise residue covariation analysis, we observe that E2 enzymes have a set of residues that vary as a group throughout evolution. Extensive mutagenesis of the E2 enzyme UbcH5b, including its covarying residues, yield mutants that can accept ubiquitin, break the thioester linkage, bind E3 enzymes, but cannot be stimulated to release their ubiquitin thioesters in the presence of an E3 enzyme. We also show that thioester release measurements agree with polyubiquitination of physiological targets. We have structurally characterized our model E2 enzyme UbcH5b, it mutants, and its interaction with E3 enzymes. Taken together, our data strongly demonstrate that E3 enzymes are, apart from their scaffolding function, activators of E2 enzymes to ubiquitinate targets. We discuss possible physical mechanisms of this activation, and hypothesize a long-distance communication pathway between the E3-binding surface and the thioester-forming active site of E2 enzymes. We also report attempts to crystallize several E2 and E3 enzymes and their complexes.Item Modulation of Transcription Factor Activity by Mono-ubiquitin(2008-09-12) Archer, Chase Tanner; Kodadek, ThomasThe Ubiquitin-Proteasome Pathway plays both proteolytic and non-proteolytic roles in the regulation of transcription. We recently reported that the ATPases of the 26S proteasome can destabilize activator-DNA complexes in a non-proteolytic manner that requires direct interactions between the Rpt4 and 6 subunits with the activation domain of the activator. Remarkably, mono-ubiquitylation of the activator blocks this repressive activity. In this study, we probe the mechanism of this protective effect. Using novel label transfer and chemical cross-linking techniques, we show that ubiquitin contacts the ATPase complex directly, apparently via Rpn1 and/or Rpt1, and that this interaction results in the dissociation of the activation domain-ATPase complex via an allosteric process. We also provide in vivo evidence demonstrating the importance of monoubiquitylation in inhibition of activator-DNA destabilization. A model is proposed in which activator mono-ubiquitylation serves to limit the lifetime of the activator-ATPase complex interaction and thus the ability of the ATPases to unfold the activator and dissociate the protein-DNA complex.Item A regulatory mechanism for Rsp5, a multifunctional ubiquitin ligase in Saccharomyces cerevisiae: characterization of its interaction with a deubiquitinating enzyme(2006) Kee, Younghoon; Huibregtse, Jon M.HECT E3 ubiquitin ligases are widely distributed from yeast to human cells and play important roles in many physiological processes. Rsp5, an essential HECT E3 ligase in Saccharomyces cerevisiae, is involved in many biological processes, including transcriptional activation, endocytic trafficking, mitochondrial inheritance, and RNA export pathways. Although Rsp5 has been shown to regulate multiple pathways targeting multiple substrates, mechanisms for regulating the biochemical activity of Rsp5 are largely uncharacterized (121, 199). To gain further insight into the regulation of this enzyme, I identified proteins that copurified with epitope-tagged Rsp5. Ubp2, a deubiquitinating enzyme, was a prominent copurifying protein. Rup1, a previously uncharacterized UBA domain protein, was required for binding of Rsp5 to Ubp2 both in vitro and in vivo. Biochemical and genetic evidence are consistent with a model that Ubp2and Rup1 antagonizes Rsp5-catalyzed substrate ubiquitination. In vivo and in vitro experiments showed that Rsp5 and Ubp2 display strong preferences for assembly and disassembly of K63-linked polyubiquitination, respectively. A large fraction of the K63 conjugates in ubp2∆ cells bound to Rsp5, and a proteomics approach was therefore used to identify Rsp5 substrates subject to Ubp2 regulation. Two proteins implicated in cell wall integrity, Csr2 and Ecm21, were identified and both proteins were efficiently K63- polyubiquitinated by Rsp5 and deubiquitinated by Ubp2. I have also shown that cell wall integrity is impaired in rsp5-1 cells and this can be rescued by either ubp2∆ or rup1∆ mutation, suggesting that the Ubp2/Rup1 complex negatively regulates Rsp5-mediated cell wall homeostasis. Together, these data represent a novel regulatory mechanism for Rsp5 and suggest that similar mechanisms might be utilized by its mammalian homologues. Furthermore, this work provides a basis for studying the mechanism for differential polyubiquitin chain type synthesis by HECT E3 ligases.Item Rules of helix termination by the c-capping box: calorimetric and spectroscopic studies(Texas Tech University, 1999-05) Thomas, Susan TanseyInterest in increasing the reliability of protein structural prediction has focused attention on exploring mechanisms of stabilization of helical domains by comparing the helical forming potential of each amino acid residue at different positions in the a-helix. To achieve this, detailed knowledge of thermodynamic information of protein stability with different sequences and under different solvent conditions is required and must be combined with information about the structural properties of these proteins. Using differential scanning calorimetry, circular dichroism, and fluorescence and NMR spectroscopy combined with site directed mutagenisis and chemical modifications of selected positions on the protein sequence, we studied several model systems. The ionic composition of a protein's environment is very important for its function and stability. At this time there is no common understanding of the role neutral salts play in the stabilization and destabilization of proteins. In order to address this, we studied the effects of neutral salts on the stability of the ubiquitin molecule. We found that the stability of the ubiquitin molecule increases with an increase in the concentration of electrolytes. This increase correlates with anion concentration rather than with an increase in ionic strength. This strongly suggests that the stabilizing effect occurs via specific bhiding of anions. We found an interesting interpretation of this. GdmHCl, a well known protein denaturant, can have dual actions on ubiquitin, the classical denaturant effect from the Gdm+ and a stabilizing effect from the CI. We also have complete thermodynamic analysis of more than 50 single and double, natural and non-natural amino acid substitutions at positions C-cap and C4-C" in the C-capping box of the a-helix in the ubiquitin molecule. We found that all of these variants of ubiquitin have perturbed thermodynamics of folding. From this we have generated a propensity scale of amino acid residue substitutions at the C-cap position In substitutions at positions C4 and C there were several cases in which we observed changes in the enthalpy and heat capacity of unfolding. A decrease in the enthalpy of unfolding and an increase in the heat capacity change upon unfolding is corroborated by the observation of partial cold denaturation of some of the hydrophobic variants of ubiquitin.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 Ubiquitin Mediated Regulation of NF-KB Signaling(2008-05-13) Pineda, Gabriel; Chen, Zhijian J.NF-κB signaling is involved in many vital cellular functions such as immunity, cell proliferation, inflammation, and apoptosis. The activation of NF-κB signaling requires the process of ubiquitination. K63-and K48-linked ubiquitin chains have been shown to have distinct roles and biological function in NF-κB signaling. K63-linked ubiquitin chains are required for the activation of TAK1, which leads to the activation of IKK. Activation of IKK leads to K48-linked ubiquitination, and the subsequent proteasomal degradation of IκBalpha . Two important areas of research focusing on ubiquitin regulation of NF-κB signaling are addressed in this dissertation. The areas addressed include understanding how ubiquitinated substrates are targeted for proteasomal degradation and how CYLD negatively regulates NF-κB signaling. In these studies, I investigated the molecular mechanisms involved in the regulation of IκBalpha degradation. Using a siRNA approach, NPL4 was shown to be required for IκBalpha degradation. In vitro proteasomal degradation assays demonstrated that the NPL4 complex is required for IκBalpha degradation. Evidence from both in vitro and in vivo studies suggest NPL4 is required for IκBalpha degradation, but not for IKK activation. These results suggest NPL4 is working at a step after ubiquitination of IκBalpha , but before proteasomal degradation. I propose that ubiquitinated IκBalpha is targeted to the proteasome by an interaction between the NPL4 complex that is mediated through the zinc finger domain of NPL4. The cylindromatosis tumor suppressor gene (CYLD) encodes a 110 kDa deubiquitination enzyme that negatively regulates NF-κB signaling. Loss-of-function mutations in CYLD lead to the disease Familial Cylindromatosis, which is characterized by the formation of benign skin tumors that originate from the head and neck of individuals afflicted with the disease. Here I present in vitro evidence that CYLD inhibits both TAK1 and IKK activation by TRAF6 in a cell free system. I also demonstrate, using a highly purified in vitro system, that CYLD specifically cleaves K63 linked ubiquitin chains and harbors endoproteolytic activity. Furthermore, the third CAPGLY domain of CYLD was shown to be a novel ubiquitin binding domain. My results provide biochemical evidence that CYLD functions as a K63 deubiquitinase to attenuate NF-κB signaling.Item Ubiquitin, the Proteasome, and Dynamics at the Protein/DNA Interface(2006-05-16) Nalley, Kip A.; Kodadek, ThomasRecently it has been discovered that a mutant species of Gal4, that contains a three amino acid change in a surface loop of the DNA binding domain, does not occupy the GAL 1/10 promoter under Gal4 inducing conditions as measured by Chromatin Immunoprecipitation (ChIP) assays. However, this protein, Gap71, occupies the promoter similarly to Gal4 under non-inducing (poised) conditions. Additionally this protein was found to be poorly ubiquitylated in vitro under conditions where Gal4 is ubiquitylated. In order to determine the mechanisms involved in the protein destabilization I have examined the properties of the individual mutations that comprise Gap71. These experiments have revealed that serine 22 is a site of phosphorylation of the Gal4 DBD and that lysine 23 is essential for S22 phosphorylation, possibly acting as part of the kinase recognition site. Mutation of either residue blocks Gal4 DBD phosphorylation, its subsequent ubiquitylation and compromises the ability of the activator to bind promoter DNA in vivo. These data represent the first report of an essential phosphorylation event for this paradigmatic transcription factor. In addition, experiments were done to directly measure the dynamics of the Gal4 /DNA complex. To measure the dynamics I have exploited the system developed by Dr. D. Picard and others using the Gal4 DNA binding domain fused to the estrogen receptor ligand binding domain. Each of these constructs has been shown to be inactive until the addition of estradiol, when they are released and bind the Gal4 UAS. These constructs allow me to temporally control the appearance of a large quantity protein that is able to compete with the endogenous Gal4 for the UAS sites in the genome. Under non-inducing conditions, the results are consistent with a rapidly exchanging complex. However, upon induction, the Gal4-promoter complexes "lock in" and exhibit long half-lives of one hour or more. Furthermore, pharmacological inhibition of proteasome-mediated proteolysis had little or no effect of Gal4-mediated gene expression. These studies show that proteasome-mediated turnover is not a general requirement for transactivator function and, when considered in the context of previous studies, that different transactivator-promoter complexes can have widely different lifetimes.