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, Marc C.The 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 ε Subunit of the T Cell Receptor(2008-05-13) Watts, Laura Michelle; van Oers, Nicolai S. C.The 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, Joseph P.Dynamins 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 Functional Significance of Extracellular Signal Regulated Kinase (ERK2) Phosphorylation States: Implications for DNA Binding(2013-11-04) McReynolds, Andrea Christine; Rice, Luke M.; Cobb, Melanie H.; Albanesi, Joseph P.; Yu, GangThe protein kinase extracellular signal-regulated kinase 2 (ERK2) has been well understood structurally for nearly twenty years. New insight is emerging about its structure and function. A novel autophosphorylation has recently been found to occur on a critical active site residue, threonine 188. Autophosphorylation of this residue has been suggested to occur as a result of the confluence of receptor tyrosine kinase (RTK) and G protein-coupled receptor (GPCR) signaling pathways. The possibility of autophosphorylation on threonine 188 seemed to be inconsistent with what was known about the structure and function of protein kinases generally and ERK2 in particular; T188 and the comparable residue in other protein kinases is required for catalytic activity. I found ERK2 phosphorylated on T188 in vitro in partially purified preparations of the recombinant protein purified following expression in bacteria. This suggests that phosphorylation of ERK2 on T188 can occur without the input of upstream RTK or GPCR signaling. Through mutagenesis experiments, I found mutation of T188 sharply reduced activity toward substrates in vitro. Protein fractions containing pT188 and purified ERK2 T188D and T188E mutants, that may act as phosphomimetics, appear to have an increased affinity for DNA binding. I examined critical residues in the activation loop important for phosphorylation and activation of ERK2 and found that perturbation of these residues influences DNA binding specificity. Although the MAPK pathway and role of ERK2 is well understood, our data suggest that previously unrecognized, higher-order signaling mechanisms that arise from additional phosphorylation events may be involved in less well characterized properties of ERK2. Second, our finding that T188 autophosphorylation can occur in recombinant ERK2 independently from upstream GPCR signaling illustrates the need for a reinvestigation of the regulation of ERK2 autophosphorylation. Finally, direct and specific DNA binding may be driven by differences in phosphorylation. The result of this work serves to redefine a most important signaling molecule in terms of its structural modifications and relationship to overall function.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 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 Novel Insights into DNA Double-Strand Break Repair and Its Cancer Implications(2016-07-27) Hardebeck, Molly Catherine; Shay, Jerry W.; Brekken, Rolf A.; Bachoo, Robert; Burma, SandeepDespite the aggressive treatment with DNA damage-inducing agents, glioblastomas (GBM) inevitably develop therapy resistance, leading to relapse and patient mortality. Cancer cells that survive therapy acquire additional damage-induced oncogenic changes that likely facilitate therapy resistance and tumor recurrence. To understand which damage-induced oncogenic alterations may promote tumor recurrence, we previously irradiated brains of mice harboring deletions of key tumor suppressors frequently lost in GBM. The most significant acquired alteration was amplification of the Met tyrosine kinase. We find that Met-expressing cells display cancer stem cell properties, augmented tumorigenesis, up-regulation of numerous DNA damage response (DDR) proteins, and an extended G2/M arrest. We hypothesize that Met expression drives therapy resistance and may be a potential target for radiosensitizing GBM. An alternative sensitization approach could involve direct inhibition of key DDR proteins, specifically in the homologous recombination (HR) double-strand break (DSB) repair pathway which is implicated in radioresistance of GBM stem cells. One indispensable step of HR is DNA-end resection, primarily executed by the exonuclease EXO1. We found that an EXO1 construct lacking the C-terminus and containing only the nuclease domain does not localize to DSBs, causing severe resection and repair defects. We hypothesized that the C-terminus of EXO1 serves as a platform for proteins to regulate EXO1's function. We found that the C-terminus interacts with BLM helicase, and it contains four Ser/Thr-Pro sites that are phosphorylated by CDKs1/2 to promote resection. We are currently examining whether CDK phosphorylation of EXO1 modulates the duration of the G2/M checkpoint since proper DNA repair requires a halt in the cell cycle. We are using CRISPR technology to generate EXO1 knock-out cells that will be complemented with WT or CDK-mutant EXO1 for checkpoint studies. We hypothesize that CDK phosphorylation of EXO1 serves to regulate resection and sustain the G2/M checkpoint. To further elucidate the role of EXO1 in maintaining genomic stability, we examined a cancer-associated SNP in EXO1 and found that it causes resection and DSB repair defects which may contribute to genomic instability and cancer progression. Overall, we provide novel insights into multiple aspects of DSB repair and identify potential targets for cancer therapy.Item The Regulation of Autophagy and Its Role in Mitotic Exit(2014-07-14) An, Zhenyi; Tu, Benjamin; Cobb, Melanie H.; White, Michael A.Autophagy is an evolutionarily conserved pathway in which cells enclose cytoplasmic contents in double membrane vesicles and deliver them to the lysosome for degradation. Autophagy plays critical regulatory roles in cancer, aging, neurodegeneration, immunity and many other physiological processes. Autophagy can be induced by multiple conditions such as starvation, viral infection, exercise and oxidative stress. In this work, we studied the role of autophagy in starvation-induced cell cycle arrest and quiescence entry, and studied the function and regulation of a major phosphorylation site of Beclin 1, serine 90. In the first study, we found that the in response to starvation, autophagy-deficient yeasts failed to arrest properly in G1/G0, but arrested in telophase with a quiescent-specific phenotype. In a second study, we found that Beclin 1 serine 90 is a major phosphorylation site of Beclin 1, which is induced by multiple stresses such as starvation and osmotic stress. The phosphorylation of Beclin 1 serine 90 leads to the activation of autophagy and inhibition of tumorigenesis. We identified MK2/3 as kinases that positively regulate autophagy by phosphorylating Beclin 1 at amino acid residue serine 90. We also found that Beclin 1 serine 90 phosphorylation is negatively regulated by Bcl-2 and positively regulated by AMPK. Beclin 1 serine 90 phosphorylation is also important for cell survival during high osmotic stress. Taken together, these results suggest that Beclin 1 serine 90 phosphorylation is a critical event in autophagy induction, which is tightly regulated by multiple kinases and regulatory proteins.Item Signal Specific Ubiquitination and Degradation of IkBa(2003-10-08) Hakala, Kevin William; Kodadek, Thomas J.The 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 Spindle Checkpoint at Kinetochores(2014-07-24) Kim, Soonjoung; Sternweis, Paul C.; Cobb, Melanie H.; Rice, Luke M.; Yu, HongtaoThe kinetochore—a large protein assembly on centromeric chromatin—functions as the docking site for spindle microtubules and as a signaling hub for the spindle checkpoint. The Constitutive Centromere-Associated Network (CCAN) at the inner kinetochore nucleates the formation of the mature outer kinetochore during mitosis, including the recruitment of the KMN network that consists of Knl1, the Mis12 complex (Mis12C), and the Ndc80 complex (Ndc80C). The KMN is a critical receptor for microtubules, and provides a landing pad for various spindle checkpoint proteins and regulatory factors. The spindle checkpoint protein Mad2 has multiple conformations, including the inactive open Mad2 (O-Mad2) and the active closed Mad2 (C-Mad2). The kinetochore-bound checkpoint protein complex Mad1–Mad2 promotes the conformational activation of O-Mad2 and serves as a catalytic engine of checkpoint signaling. The activated C-Mad2 binds to and inhibits Cdc20, an activator of APC/C, to prevent precocious anaphase onset. Deficient spindle checkpoint signaling leads to premature sister-chromatid separation and aneuploidy. Research in this thesis has provided several key insights into spindle checkpoint signaling at kinetochores. First, we show that the conformational transition of Mad2 is regulated by phosphorylation of S195 in its C-terminal region. The phospho-mimicking Mad2S195D mutant and the phospho-S195 Mad2 protein do not form C-Mad2 on their own. Mad2 phosphorylation inhibits its function through differentially regulating its binding to Mad1 and Cdc20. Our results establish for the first time that the conformational change of Mad2 is regulated by posttranslational mechanisms. Second, we have studied how Mad1 is targeted to kinetochores. We have determined the crystal structure of the conserved C-terminal domain (CTD) of human Mad1. The structure reveals unexpected fold similarity between Mad1 CTD and known kinetochore-binding modules. Functional studies then validate a role of Mad1 CTD in kinetochore targeting and implicate Bub1 as its receptor. Interestingly, deletion of the CTD does not abolish Mad1 kinetochore localization. Non-overlapping Mad1 fragments retain detectable kinetochore targeting. Our results indicate that the CTD–Bub1 connection is one of several mechanisms of targeting Mad1 to kinetochores. Finally, we show that the proper assembly of KMN is required for generating the spindle checkpoint signal at kinetochores. We have developed several strategies to inactivate KMN at kinetochores in human cells, and demonstrate its requirement for the spindle checkpoint in the absence of microtubules. We further show that two quasi-independent pathways mediate the mitosis-specific assembly of KMN at kinetochores. In one pathway, the centromeric kinase Aurora B phosphorylates the Mis12C component Dsn1, and strengthens Mis12C binding to the CCAN component CENP-C. In the second pathway, CENP-T anchors the CENP-H/I/K sub-complex at kinetochores, which in turn recruits Ndc80C. Inactivation of both pathways abolishes KMN at kinetochores and causes gross spindle checkpoint defects. In conclusion, combining cell biology and structural biology methods, our studies have defined a new posttranslational mechanism of Mad2 regulation, uncovered a critical way for targeting Mad1 to kinetochores, and dissected assembly pathways of the KMN checkpoint sensor at kinetochores.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.