Browsing by Subject "Protein-Serine-Threonine Kinases"
Now showing 1 - 20 of 20
Results Per Page
Sort Options
Item Analysis of Aurora B Regulation and Signaling(2006-05-16) Oncel, Dilhan; Yu, HongtaoAurora B is a serine/threonine kinase that functions in a complex with two other chromosomal passenger proteins called INCENP and Survivin. Its function is implicated in a variety of processes related to mitosis, such as chromosome condensation, regulation of arm cohesion, spindle assembly, chromosome bi-orientation and cytokinesis. During the cell cycle, the level of this protein is tightly controlled and its deregulated abundance is suspected to contribute to aneuploidy. The cell cycle profile for Aurora B is reminiscent of those for substrates of the anaphase-promoting complex/cyclosome (APC/C), an ubiquitin ligase essential for mitotic progression. Here, we showed that Aurora B is a substrate of APC/C both in vitro and in vivo. Aurora B is efficiently ubiquitinated iv in an in vitro reconstituted system by APC/C that had been activated by Cdh1. The recognition of Aurora B by APC/CCdh1 is specific as it requires the presence of a conserved KEN-box motif at the amino terminus of Aurora B. Degradation of Aurora B at the end of mitosis requires Cdh1 in vivo as the reduction of Cdh1 level by RNA interference stabilizes Aurora B protein. We conclude that, as a key mitotic regulator, Aurora B is degraded by APC/CCdh1 in late mitosis. Aurora B lies at the heart of the cellular mechanism that resolves synthelic and merotelic attachments. A failure to eliminate such events results in gain or loss of chromosomes. Therefore, identifying the physiological substrates of Aurora B is of pivotal importance for research. We screened Aurora B substrates using an in vitro expression cloning system. However, the methodology we employed didn't lead to candidate substrates to be further validated by more rigorous in vivo approaches. The use of high concentrations of misfolded recombinant Aurora B was partially responsible for the loss of specificity. Therefore, purifying active recombinant Aurora B has become a primary goal for future biochemical and structural work. Two molecular chaperones Hsp90 and Cdc37 assist the folding of a variety of kinases in vivo, among which Aurora B is also a candidate. This gave us the final idea of expressing Aurora B-INCENP complexes in bacteria via the coexpression of Hsp90-Cdc37 molecular chaperones.Item Autoinhibition and Chloride Sensing in the WNK1 Kinase(2012-07-16) Moon, Thomas Matthew; Goldsmith, ElizabethProtein kinases control diverse cellular pathways and have are the subject of intensive study regarding how they maintain specificity toward sub- strates. The research presented here focuses on a 230kDa serine/threonine protein kinase known as WNK1 (with no lysine {k}). The protein was first cloned by Melanie Cobb’s laboratory, and its isoforms have been associated with a monogenic form of hypertension as well as with breast and prostate cancer. Recent data have also shown that WNK1 is necessary for maintaining spindle polarity in mitosis and plays a role in post-mitotic abscission. The function of WNK1 is most commonly associated with the regulation of CCCs via the activation of the WNK1 substrates OSR/SPAK. Prior investigation of the system has demonstrated that CCCs are activated by increasing con- centrations of extracellular salt and by intracellular phosphorylation from the OSR/SPAK kinases. Due to it ubiquity in mammalian cell types, a question has arisen as to how the pathway responds to changes in osmolarity. Further, because of the involvement of WNK1 in a diverse set of cellular mechanisms, how is WNK1 activity and substrate specificity controlled? An autoinhibitory domain of WNK1 was characterized by the Cobb Lab regarding its ability to inhibit the kinase in cis and in trans. In this study, we find that the solution structure of the autoinhibitory domain retains a conserved RFXV binding site from the PASK/FRAY homology 2 (PF2) domain present in OSR/SPAK. Titration data shows that incubation with a 5-mer and a 20-mer peptide derived from the WNK1 kinase domain displays extensive chemical shift perturbation as assessed by 1H,15N-HSQC. Expression of this autoinhibitory domain in cis with the WNK1 kinase domain followed by size-exclusion chromatography shows substantial confor- mational changes when dialized from high to low salt. A measurement of the activity of the WNK1 kinase domain in the presence of increasing amounts of sodium chloride indicate an IC50 of 130mM. Further biophysical investigation using differential scanning fluorimetry with the kinase domain shows that the domain undergoes substantial increases in domain stabilization as the concen- tration of salt is increased. Continued analysis of this phenomena has pointed toward evidence of anion sensing by the WNK1 kinase domain. Other protein kinases studied in our lab do not exhibit this salt sensitivity. To determine the binding site of chloride in the WNK1 kinase domain, the inactive WNK1 kinase was cocrystallized in the presence of sodium bro- mide. A dataset was collected using the bromine anomalous edge (0.92 ̊A). The anomalous difference fourier map was calculated and a 5.2 σ peak was observed at the N-terminus of the 3.10 helix present in the DLG motif of the activation loop. To corroborate these data, the structure of the inactive kinase domain previously crystallized in sodium chloride was re-refined. A similar binding site corroborated by a 2mFo − DFc peak of 5.5 σ was observed in subunit A near the N-terminus of the 3.10 helix. When the structure was refined in with a chloride ion placed in the observed density, similar hydrogen bonding interactions between the amide backbone and the chloride ion were observed compared to that in the bromide-soaked structure. The presence of this chloride ion appears to favor sequestration of E268 in αC and R348 in the catalytic loop and promotes an inactive kinase structure. Finally, the crystal structure of the activated WNK1 kinase domain was determined under low-salt conditions. The term ’activated’ and not active is used to describe this structure of the WNK1 kinase domain because, although it is phosphorylated at S378 and S382 in the activation-loop during expression, the structure adopts an inactive conformation due to the placement E268 in helix C. The structure displays disorder of many key structural elements such as the N-terminus of αC. A key observation is the lack of a 3.10 helix in the N-terminus of the activation loop and the lack of water or any atom that could be chloride near the amide backbone near the chloride binding site. Based upon the literature surrounding the activation of WNK1 and the data presented in this thesis, we predict a three-tiered regulation of WNK1 driven by a) autophosphorylation b) chloride binding and c) autoinhibitory domain occlusion of the nucleotide and/or docking interfaces present in the WNK1 kinase domain. The coupling of the information that we have gath- ered on the autoinhibitory and kinase domains appear to point to an overall mechanism of salt sensing and self-contained signaling control in the WNK1 kinase cascade. [Keywords: WNK1, transferase, autoinhibition, chloride-sensing, PF2, kinase]Item Dissection of Mechanisms Regulating the Drosophila Hedgehog Pathway(2012-07-20) Shi, Qing; Jiang, JinHedgehog (Hh) signaling is essential for both embryonic development and adult tissue homeostasis. Malfunction of Hh signaling pathway causes many human disorders including birth defects and cancers. In Drosophila, the G-protein-coupled-receptor-like protein Smoothened (Smo) transduces the Hh signal across the plasma membrane, and an intracellular Hh signaling complex (HSC) containing the kinesin-related protein Costal2 (Cos2), the serine/threonine protein kinase Fused (Fu) and a PEST-domain containing protein suppressor of Fused (Sufu) relays the Hh signal downstream from Smo to the Zn finger transcription factor Cubitus interruptus (Ci). Our previous studies have demonstrated that Hh transduces signal by regulating the subcellular localization and conformational state of Smo, but how Smo relays the signal to cytoplasmic signaling components remains poorly understood. In this study, we show that Hh-induced Smo conformational change promotes the recruitment of Cos2/Fu complex and Fu dimerization. We find that induced dimerization through the Fu kinase domain activates Fu by inducing multi-site phosphorylation of its activation loop (AL), and phospho-mimetic mutations of AL suffice to activate the Hh pathway. Moreover, we find that activated Fu regulates Ci by both promoting its transcriptional activator activity and inhibiting its proteolysis into a repressor form. We provide evidence to suggest that activated Fu exerts the regulation by interfering with the formation of Ci-Sufu and Ci-Cos2-kinase complexes that normally inhibit Ci activity and promote its processing. In the rest part of the study, we further explore additional mechanisms regulating Ci activity. We have identified and characterized three types of functional regulatory elements in Ci, including a transcriptional repression domain in the N-terminal region of Ci, multiple Ser/Thr motifs in the amino-(N-) and carboxy-(C-) terminal regions of Ci serving as HIB/SPOP E3 ligase-specific degrons, and finally a novel PY-NLS around the N-terminal highly conserved domain of Ci. [Keywords: hedgehog, Drosophilia, Fu, Cos2, Ci, Sufu, Smo, HIB, PY-NLS]Item Evaluation of Chronic RalGTPase Activation as a Core Specifier of Oncogenic Transformation(2009-01-08) Cheng, Tzuling; White, MichaelRal (RAS-Like) GTPases, RalA and RalB, were originally identified based on sequence similarity to Ras and are directly activated via the Ras effector family Ral guanine nucleotide exchange factors (RalGEFs). Previous studies have demonstrated that RalA and RalB collaborate to maintain tumorigenicity through regulating both proliferation and survival. Remarkably, RalB is specifically required for survival in Ras-dependent tumor cells rather than normal cells, while RalA is required for anchorage-independent proliferation but dispensable for survival. However, the spectrum of cancer cell lineages dependent upon Ral functions for tumor formation is currently unknown. We examined whether Ral pathway activation is required for proliferation of cancer cells with activated Ras, Raf, or PI3K. Our data indicate that the Ral pathway is aberrantly activated and required for maintaining tumorigenicity of cancers that are driven by oncogenes other than Ras. In order to begin to understand how the Ral pathway may be chronically engaged in diverse oncogenic backgrounds, we further examined the expression of RalGEFs in a variety of cells derived from different tissue origin. Our results showed a divergent and complex distribution of RalGEFs among different cell types. In addition, through examination of historical tumor resequenceing efforts, we found several somatic mutations in RalGEFs, including RalGDS and RGL1. Through biochemical and cell biological studies, we find that the RGL1 mutations identified in human breast cancers are gain-of -function mutations, and found the mutations contribute to tumor cell survival through RalB pathway. Furthermore, we showed that chronic activation of RGL1 is sufficient to transform immortalized human mammary epithelial cells. Together, our data suggest RGL1 is a bona fide oncogene. These studies broaden our knowledge about RalGEF-Ral contributions in tumorigenicity, and provide a potential target for cancer therapeutic interventions.Item Functional Genomics Based Interrogation of Cell-Fate Determination Pathways(2011-08-26T17:34:56Z) Jacob, Leni Susan; Lum, LawrenceThe Hedgehog (Hh) and Wnt signal transduction pathways are master regulators of embryogenesis and tissue renewal and represent anticancer therapeutic targets. Using genome-wide RNA interference screening in murine cultured cells, I established previously unknown associations between these signaling pathways and genes linked to developmental malformations, diseases of premature tissue degeneration, and cancer. I identified functions in both pathways for the multitasking kinase Stk11 (also known as Lkb1), a tumor suppressor implicated in lung and cervical cancers. Stk11 loss resulted in disassembly of the primary cilium, a cellular organizing center for Hh pathway components, thus dampening Hh signaling. Loss of Stk11 also induced aberrant signaling through the Wnt pathway. Chemicals that targeted the Wnt acyltransferase Porcupine or that restored primary cilia length by inhibiting the tubulin deacetylase HDAC6 (histone deacetylase 6) countered deviant pathway activities driven by Stk11 loss. My study demonstrates that Stk11 is a critical mediator in both the Hh and the Wnt pathways, and that functional genomics based approaches to dissecting cell-fate determination pathways may support the development of targeted therapeutic strategies.Item The Hippo Signaling Pathway in Organ Size Control and Regeneraton(2012-07-17) Ren, Fangfang; Jiang, JinThe Hippo (Hpo) signaling pathway controls cell growth, proliferation and apoptosis in both Drosophila and vertebrates. Our lab has previously demonstrated that Hpo signaling regulates gene expression by inhibiting a transcription complex consisting of the transcriptional coactivator Yorkie (Yki) and the TEAD/TEF family of transcription factor Scalloped (Sd) in Drosophila. The inhibition of Yki activity is through modulating its phosphorylation status and subcellular localization by upstream kinase complex. I obtained both genetic and cellular evidence that 14-3-3 proteins are involved in this process. I also identified three Serine residues (S111, S168 and S250 of Yki as essential for restricting Yki activity. I found that 14-3-3 regulates Yki subcellular localization mainly through S168 but not the other two sites. The recent identification of intestinal stem cells (ISCs) has made the Drosophila adult midgut an excellent model to study adult stem cell biology. Multiple signaling pathways have been implicated in the regulation of ISC proliferation, self-renewal and differentiation. I obtained evidence that Hpo signaling plays an essential role in regulating ISC proliferation through both cell-autonomous and non-cell-autonomous mechanisms. Cytokines of the Upd family and multiple EGFR ligands were found to be ectopically induced when Hpo signaling is inactivated in differentiated cells, which in turn activate Jak-Stat and EGFR signaling pathways in ISCs to stimulate their proliferation. I also showed that tissue damaging reagent DSS-induced ISC proliferation is dependent on Yki activity in precursor cells. Although several signaling pathways including Jak-Stat, EGFR and Hpo pathways have been implicated in damage-induced ISC proliferation, the cell intrinsic mechanisms have remained elusive. I found that the Drosophila homolog of Myc oncogene (dMyc), which encodes a transcription regulator that affects cellular growth and cell cycle progression, functions downstream of Hpo, Jak-Stat and EGFR pathways to mediate their effects on ISC proliferation. dMyc is also essential for adult midgut homeostasis as well as regeneration after exposure to damage reagents. I also demonstrated that the regulation of dMyc levels by Hipo, Jak-Stat and EGFR pathways is at the level of transcription. [Keywords: Drosphila, hippo, Yki, instestine stem cell, regeneration]Item Identification of Substrates and Pathways Regulated by PAS Kinase(2005-12-20) Probst, Brandon Linn; McKnight, StevePAS kinase, a serine/threonine protein kinase, is unique in that it comprises the only mammalian protein kinase regulated by a PAS domain. The interest of the McKnight laboratory in understanding the regulation and biological role of PAS kinase stems from knowledge that PAS domains typically function as sensors in other systems. My study primarily focused on unveiling physiological PAS kinase (PASK) substrates and interacting gene products to establish pathways regulated by PASK. First, we examined the function of PASK in the budding yeast, S. cerevisiae. An unbiased biochemical screen for putative PASK substrates from cellular extracts uncovered five polypeptides phosphorylated in a PASK-dependent manner. Two of the substrates identified are known translation factors and a third is an RNA-binding protein that was also found to be a high copy suppressor of the psk1 psk2 double mutant phenotype. We further observed PASK to phosphorylate two enzymes in the pathway for the synthesis of glycogen: UDP-glucose pyrophosphorylase and glycogen synthase. Genetic and biochemical data provide evidence that both of these enzymes are inhibited by PASK-dependent phosphorylation. We next examined the role of PASK in mammalian cells. With the exception of glycogen synthase none of the mammalian homologs of the yeast PASK substrates have been found to qualify as substrates for the mammalian PASK. To this end, a second unbiased, large-scale biochemical screen was employed using HeLa cell extracts to discover phosphorylation targets of the mammalian enzyme. This biochemical screen entailed the disruption of 150 liters of HeLa cells into a soluble extract and subsequent fractionation over seven chromatographic steps to generate roughly 1000 partially purified pools of protein. This effort led to the identification of enzyme substrates involved in protein synthesis and intermediary metabolism. Phosphorylation site mapping of these substrates identified the consensus motif, R-X-A/x-S*/T* as the optimal substrate for PASK. Although it remains unclear as to the functional role of PAS kinase-dependent phosphorylation of these substrates, there appears to be a unifying theme in both yeast and mammalian systems. PAS kinase is a PAS-domain regulated enzyme controlling translation as this energetically expensive process is coupled to cellular energy metabolism.Item Identification of Substrates and Pathways Regulated by WNK1(2004-12-15) Lee, Byung-Hoon; Cobb, Melanie H.WNK (With No lysine (K)), a serine/threonine protein kinase, is a unique molecule not belonging to any other canonical protein kinase family including mitogen-activated protein (MAP) kinases. The name of the WNK protein kinase family reflects the fact that a catalytic lysine lies in a position different in WNKs from that in all other protein kinases. The urgency of a mechanistic examination of the WNK family protein kinase was heightened by the discovery that mutations in at least two of the four human WNKs, WNK1 and 4, caused a heritable form of hypertension. My study focused on unveiling WNK1 substrates and interactors for a better understanding of the molecular pathways served by WNK kinases. Yeast two-hybrid screening was performed to identify the binding partners of WNK1 and yielded genuine interactors including synaptotagmin (Syt) isoforms, Smad2, and dynein light chain (LC8/PIN). WNK1, not WNK4, selectively binds to and phosphorylates Syt2 within its calcium binding C2 domains. Calcium strongly enhanced their binding in vitro. Essential Ca2+-binding residues in the Syt2 C2 domains were critical for formation of a WNK1-Syt2 complex and for Syt2 phosphorylation. WNK1 displayed specificity among Syt isoforms and mutational analysis implicated a hydrophobic residue on the WNK1 kinase domain surface as essential for the high affinity WNK1-Syt2 interaction and phosphorylation. Endogenous WNK1 and Syt2 coimmunoprecipitated and colocalized on a subset of secretory granules in the INS-1 cell line, a pancreatic beta cell model system. Importantly, phosphorylation by WNK1 increased the amount of Ca2+ required for Syt2 binding to phospholipid vesicles; mutation of Thr202, a WNK1 phosphorylation site identified from mass spectrometric analysis, partially prevented this change. These findings provide a biochemical scenario that could lead to the retention or insertion of proteins in the plasma membrane. WNK1 may serve as a molecular switch for vesicle trafficking and other membrane events that regulate ion balance. The interaction with and phosphorylation of other molecules by WNK1 were also investigated here.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 Mechanistic Studies of Autophagy Initiation in Mammalian Cells(2011-08-26T17:35:06Z) Shang, Libin; Wang, XiadongMacroautophagy (herein referred to as autophagy) is an evolutionarily conserved self-digestive process cells use to adapt to starvation and other stresses. During autophagy, portions of cytoplasmic materials are engulfed into specialized double-membrane structures to form autophagosomes, which then fuse with lysosomes to degrade their cargos and regenerate nutrients. Initiation of autophagy has been extensively studied in budding yeast Saccharomyces cerevisiae. However, various significant differences exist between yeast and mammals. To pinpoint how mammalian autophagy is initiated, I first adopted proteomic approaches to identify associating partners of Unc-51-like kinase 1 (Ulk1), key initiator for mammalian autophagy. Two novel proteins, mAtg13 and Atg101, were found to interact with Ulk1 stoichiometrically. Knockdown of either mAtg13 or Atg101 led to decreased autophagy, and autophagy could be rescued with exogenous expression, suggesting the two proteins were critical for mammalian autophagy initiation. I then observed Ulk1 undergoes dramatic dephosphorylation upon starvation, particularly at serine 638 and serine 758. I found phosphorylations of Ulk1 are mediated by mammalian target-of-rapamycin (mTOR) kinase and AMP-activated protein kinase (AMPK). AMPK interacts with Ulk1 in a nutrient-dependent manner, and proper phosphorylations on Ulk1 are crucial for Ulk1/AMPK association, as a single serine-to-alanine mutation (S758A) at Ulk1 impairs this interaction. Compared to its wild-type counterpart, this Ulk1-S758A mutant initiates starvation-induced autophagy faster at early time points, but does not alter the maximum capacity of autophagy when starvation prolongs. With this layer of regulation, mammalian autophagy is capable of responding to environmental changes more promptly than previously considered.Item Regulation and Mechanism of Bub1-Mediated Spindle Checkpoint Signaling(2006-12-20) Qi, Wei; Yu, HongtaoThe spindle checkpoint is a surveillance mechanism that ensures the fidelity of chromosome segregation during mitosis and meiosis. Bub1 is a highly conserved protein serine/threonine kinase that plays multiple roles in the spindle checkpoint. The regulation and mechanism of Bub1 in spindle checkpoint were investigated. Bub1 is degraded during mitotic exit and the degradation of it is mediated by APC/C in complex with its activator Cdh1 (APC/CCdh1). Overexpression of Cdh1 reduces the protein levels of ectopically expressed Bub1 whereas depletion of Cdh1 by RNA interference (RNAi) increases the level of the endogenous Bub1 protein. Two KEN-box motifs on Bub1 are required for its degradation in vivo and ubiquitination in vitro. A Bub1 mutant protein with both KEN-boxes mutated is stable in cells. Kinetochore is the origin of spindle checkpoint signal and contains the catalytic machinery for generating the signal. We identify an ATP-dependent APC/CCdc20 inhibitory activity on metaphase chromosomes with unattached kinetochores. The Cdc20-S153A that cannot be phosphorylated by Bub1 is not inhibited by metaphase chromosomes, suggesting Bub1 is likely responsible for the inhibitory activity. Bub1 on unattached kinetochores is hyperphosphorylated and activated. Furthermore, the kinase-dead mutant of Bub1 cannot restore spindle checkpoint in Bub1-RNAi cells, demonstrating that the kinase activity of Bub1 is required for the spindle checkpoint. Plk1 is required for the generation of the tension-sensing 3F3/2 kinetochore epitope and facilitates kinetochore localization of Mad2 and other spindle checkpoint proteins. We investigate the mechanism by which Plk1 is recruited to kinetochores. We show that Plk1 binds to Bub1 in mitotic cells. The Plk1-Bub1 interaction requires the polo-box domain (PBD) of Plk1 and is enhanced by Cdk1-mediated phosphorylation of Bub1 at T609. The PBD-dependent binding of Plk1 to Bub1 facilitates phosphorylation of Bub1 by Plk1 in vitro. Depletion of Bub1 in HeLa cells by RNAi diminishes the kinetochore localization of Plk1. Ectopic expression of the wild-type Bub1, but not the Bub1-T609A mutant, in Bub1-RNAi cells restores the kinetochore localization of Plk1. Our results suggest that phosphorylation of Bub1 at T609 by Cdk1 creates a docking site for the PBD of Plk1 and facilitates the kinetochore recruitment of Plk1.Item Regulation of Endocytosis of ROMK Channel by WNK Kinase Family(2009-09-04) Wang, Hao-Ran; Huang, Chou-LongWNK kinases are members of a novel family of serine/threonine kinases with atypical placement of the catalytic lysine. Mutations in WNK1 and WNK4 cause pseudohypoaldosteronism type 2 (PHA2), an autosomal-dominant disease characterized by hypertension and hyperkalemia. Renal outer medullary potassium channel (ROMK) is responsible for constitutive K+ secretion in the kidney. WNK1 and WNK4 stimulate the clathrin-mediated endocytosis of ROMK, which contributes to the pathogenesis of hyperkalemia in PHAII patients. Intersectin (ITSN) is a multimodular endocytic scaffold protein. The proline-rich domains of WNK1 and WNK4 bind with the Src-homology domain (SH3) of intersectin, and this interaction is important for the stimulation of endocytosis of ROMK by WNKs. Intersectin will further activate the GTPase activity of dynamin and the actin polymerization of N-WASP, and thus promote endocytosis of ROMK channel. WNK1 inhibition of ROMK is further regulated by the kinase domain conformation, which is critical for WNK1 to recruit intersectin. A shorter renal alternatively spliced form of WNK1 that lacks the kinase domain, known as kidney specific WNK1 (KSWNK1),interacts with WNK1 kinase domain and antagonizes WNK1 inhibition of ROMK. The 4a domain and the auto-inhibitory domain in KS-WNK1 are responsible for the antagonization. The antagonism of WNK1 by 4a domain of KS-WNK1 can be abolished by 2-BP (a palmitoylation inhibitor) and hydrogen peroxide (generated during K+ deficiency). These results provide a molecular mechanism for the regulation of endocytosis of ROMK by WNK kinase family.Item Structural Analysis of STE20-related Proline-Alanine-Rich Kinase (SPAK)(2009-06-17) Juang, Yu-Chi; Cobb, MelanieSte20-related proline-alanine-rich kinase (SPAK) is a kinase that regulates ion cotransporters including KCC, NKCC1, NKCC2 and NCC. Recently, SPAK was identified as a target regulated by the WNK (With No lysine (K)) family of protein kinases. Overexpression of WNK1 has been associated to a hereditary form of hypertension. The link between WNK-SPAK-cotransporters provides one of the functional pathways for WNK to regulate intracellular and extracellular salt and water balance, thus contribute to the control of blood pressure. My study focused on determining the crystal structure of SPAK and providing a molecular basis for understanding the catalytic and regulatory mechanisms of this enzyme. I have solved the crystal structure of a partially active form of SPAK 63-390 (T243D). The structure contains the kinase domain and part of the C-terminal PF1 domain with a mutation of the WNK phosphorylation site, Thr243, to aspartate to mimic the phosphorylated state. The structure reveals two dimer interfaces. One is from the unique activation loop-swapped structure. The other is formed between the N-terminal domain of one molecule and the C-terminal domain of the second molecule. Comparison the structure with unphosphorylated inactive OSR1 revealed significant conformational changes in the glycine-rich loop, helix aC, and the activation loop. A more striking feature is the reformation of the P+1 pocket in SPAK. The interaction is formed by residues from both molecules of the domain-swapped dimer supporting the conclusion that the domain-swapped dimer is a functional unit. As remodeling of the P+1 pocket is usually associated with kinase activation, the conformational change found between the crystallized forms of OSR1 and SPAK is thus believed to be a regulated event. The unique activation loop-swapped feature suggests a novel regulatory mechanism that could be used in kinases.Item Structural and Functional Analysis of HIV-1 Nef Activation of PAK-2(2009-06-19) Kuo, Lillian S.; Garcia-Martinez, Victor J.Nef is an accessory protein encoded by HIV-1 that activates the host cellular p21 activated protein kinase 2 (PAK-2). Previous work has characterized the structural plasticity of Nef with regard to PAK-2 activation. Residues 89 and 191 were identified to be components of an effector domain required for Nef mediating PAK-2 activation with lesser contributions from position 85 and 188. H89 and F191 are highly conserved in subtype B Nefs (LHKF), however in subtype E Nef F89 and R191 predominate. Subtype E Nefs also activate PAK-2, therefore it appeared at least two different structural variants are present in HIV-1 Nefs. Substitution of all four residues in a subtype B Nef with subtype E-like residues (F85, F89, A188 and R191, FFAR) generated a fully functional subtype E PAK-2 effector domain in a subtype B background. A third effector domain found in subtype C Nefs (F85, F89, H188, and H191, FFHH) was also investigated. The contribution of residues 187 and 188 in these alternative Nef structural variants (LHKF, FFAR, and FFHH) to activate PAK-2 was determined. Surprisingly, the L188 substitution in the LHKF structure resulted in PAK-2 hyperactivation. While the I187 substitution in LHKF completely ablated PAK-2 activity. In stark contrast, I187 in the FFHH variant resulted in hyperactivation. Thus, subtle changes in amino acid composition can dramatically affect kinase activation levels. The work in this thesis has characterized a PAK-2 effector domain on Nef constituted by amino acid position 85, 89, 187, 188 and 191. The results indicate that this is not the only Nef region mediating PAK-2 activation. The highly conserved polyproline helix also plays a role in the activation of PAK-2. Conservative mutations of this SH3 binding region completely abrogated PAK-2 activation suggesting SH3 binding is necessary, however this binding appears to be weak. My data suggest a model where activation of PAK-2 by Nef requires a ternary, or higher order, complex containing SH3/Nef/PAK-2. Synergistic interactions between the two Nef effector domains investigated here and a host cell protein, or proteins, could explain the specific activation of PAK-2 by Nef.Item Studies of the Hippo Signaling Pathway(2012-08-13) Yue, Tao; Jiang, JinHow multicellular organisms control their growth to reach proper organ size during development is a fascinating question. Recent studies, initially from Drosophila, have identified the Hpo tumor suppressor pathway as a crucial mechanism that controls tissue growth by inhibiting cell growth, proliferation and survival. Deregulation of the Hpo pathway has been implicated in various human cancers. Central to the Hpo pathway is a kinase cassette consisting of four tumor suppressor proteins, the Ste20-like kinase Hpo, the WW domain-containing protein Salvador (Sav), the NDR family kinase Warts (Wts) and the Mob family protein Mats. The kinase activities of Hpo and Wts are facilitated by their regulatory proteins Sav and Mats, respectively. Activated Hpo/Sav complex phosphorylates and activates the Wts/Mats complex, which in turn phosphorylates and inactivates the transcriptional coactivator Yorkie (Yki). Phosphorylation of Yki restricts its nuclear localization through recruiting 14-3-3. When the activity of the Hpo/Wts kinase cassette is compromised, Yki forms complexes with transcription factors including Scalloped (Sd) and translocates to the nucleus to activate Hpo pathway target genes, including cyclin E, diap1, and the microRNA bantam that regulate cell growth, proliferation and survival. To identify novel components of the Hpo signaling pathway, I carried out a genetic modifier screen in which flies carrying GMR-Gal4 and UAS-Yki were crossed to a collection of transgenic RNAi lines from Vienna Drosophila RNAi center (VDRC) and Bloomington stock center, and looked for enhancers or suppressors of the overgrown eye phenotype caused by Yki overexpression. Through this screen, I have found that Echinoid (Ed), an immunoglobulin domain-containing cell adhesion molecule, acts as an upstream regulator of the Hpo pathway. Loss of Ed compromises Yki phosphorylation, resulting in elevated Yki activity that drives Hpo target gene expression and tissue overgrowth. Ed physically interacts with and stabilizes the Hpo-binding partner Sav at adherens junctions. Ed/Sav interaction is promoted by cell-cell contact and requires dimerization of Ed cytoplasmic domain. Overexpression of Sav or dimerized Ed cytoplasmic domain suppressed loss-of-Ed phenotypes. I propose that Ed may link cell-cell contact to Hpo signaling through binding and stabilizing Sav, thus modulating the Hpo kinase activity. Furthermore, the Cul4/WDR40A complex has also been identified as a genetic modifier for the Hippo signaling pathway. However, the exact mechanism by which this complex regulates the Hippo signaling pathway need to be further addressed. [Keywords: hippo pathway, echinoid, Salvado, cell adhesion, cell contact]Item Understanding Potassium Homeostasis Using Human and Mouse Genetic Models(2013-01-17) Cheng, Chih-Jen; Huang, Chou-Long, M.D., PH.D.Potassium homeostasis is one of the most sophisticated processes involving multiple organs in mammals. Many physiological functions, such as excitability of muscles and neurons, rely on stable extracellular potassium concentration. To maintain potassium homeostasis, endogenous factors including hormones and peptides regulate the activity of potassium transporters in many organs, especially skeletal muscles and kidneys, in response to different conditions. To study the perplexed regulations of potassium transporters, I choose two genetic models of human potassium disorders, hypokalemic periodic paralysis (hypoPP) and pseudohypoaldosteronism type II (PHA2). Patients with hypoPP are characterized with ictal hypokalemia and muscle paralysis. HypoPP can be divided into familial and non-familial forms. Recent studies have revealed the pathogenesis of familial hypoPP. However, the pathogenesis of non-familial hypoPP, mainly composed of thyrotoxic or sporadic periodic paralysis (TPP/SPP), is mostly unknown. A novel muscle-specific inward-rectifying potassium (Kir) channel, Kir2.6, has been recently suggested to play a role in TPP. Here, I focus on studying the role of Kir channels in non-familial hypoPP and propose the disease mechanisms to explain hypokalemia and muscle paralysis. PHA2 is a genetic disorder caused by mutations on with-no-lysine kinase 1 or 4 (WNK1/4) and featured with hyperkalemia and hypertension. Studies in PHA2 have revealed that WNK kinases regulate renal sodium transporters and potassium channels. WNK1 enhances the endocytosis of renal outer medullary potassium (ROMK) channel through an intersectin-dependent mechanism, but the upstream regulator is still unknown. Here, I clarified that the phosphoinositol-3-kinase-induced activation of Akt1/SGK can phosphorylate threonin 58 of WNK1 and thus inhibits ROMK current in cultured cells. In addition to full-length WNK1, the kinase-deficient WNK1 isoform, kidney-specific WNK1 (KS-WNK1), also participates in the regulation of renal sodium and potassium handling. Previous studies have shown that high potassium intake enhances KS-WNK1 expression and suppresses renal sodium reabsorption in thick ascending limb (TAL). However, the localization and function of KS-WNK1 in TAL are still debatable. Here, I used KS-WNK1 genetic mouse models to demonstrate that KS-WNK1 is present and function to inhibit sodium reabsorption in cortical TAL. These results contribute to the understanding of potassium homeostasis in skeletal muscle and kidney.Item With No Lysine (WNK) Family Proteins and Their Interaction with Downstream Kinases(2011-08-26T17:35:34Z) Wedin, Kyle Edward; Cobb, Melanie H.With no lysine (WNK) kinases are a family of protein kinases characterized by unusual kinase domain architecture. These large proteins, divergent outside of a kinase core and protein-protein interaction motifs, have been associated with pseudohypoaldosteronism 2, a form of Mendelian-inherited hypertension, and numerous downstream effectors that regulate vesicle trafficking, membrane protein localization, and ion handling. This study shows that WNK2 is also a functioning protein kinase with the same unusual kinase domain architecture and regulation by an autoinhibitory region. Like WNK1, WNK2 is able to signal to the extracellular-signal regulated kinase 5 (ERK5) pathway. One effector for WNK1 is oxidative stress responsive 1 (OSR1), a sterile20-like kinase. All four WNKs are able to phosphorylate OSR1 and stimulate its activity toward an ion transporter substrate, to roughly a similar degree. The WNKs have similar kinetic properties, with Km toward OSR1 in the micromolar range and kcat¬ near 1 min-1. No significant differences in activity toward OSR1 were seen for a mutant kinase domain at a site divergent among the WNKs that shows differential binding to substrate. Analysis of the phosphorylation sites of OSR1 reveals multiple sites along the activation loop that can promote increased activation if carrying a negative charge. It is unknown if these sites are phosphorylated in vivo. However, a second site of WNK1 phosphorylation just outside of the OSR1 kinase domain does not seem to affect WNK-OSR interactions. Further studies of interactions of the WNKs with their downstream effectors will reveal unusual functions for this unique family of proteins.Item With No Lysine 1 (WNK1): A Potential Regulator of The Lysosomal Degradation Pathway(2007-05-22) Lenertz, Lisa Yvonne; Cobb, Melanie H.With no Lysine (K) 1 (WNK1) is an atypical serine/threonine protein kinase that has its catalytic lysine positioned in a unique location. This kinase, along with another member of the WNK family, WNK4, has been genetically linked to pseudohypoaldosteronism type II (PHAII), which is characterized by both hypertension and hyperkalemia. Several groups have used reconstitution assays in Xenopus oocytes and mammalian cell lines to show WNKs regulate the surface expression and/or activity of various ion transporters and channels, including the epithelial sodium channel (ENaC) and the sodium chloride co-transporter (NCCT). Although the mechanisms for regulating these cell surface proteins are not well defined, it appears that WNKs may modulate the intracellular trafficking of these channels and transporters. To help define the mechanisms WNK1 utilizes to influence blood pressure and to characterize this kinase biochemically, I performed a WNK1 kinase activation screen and a WNK1 yeast-two-hybrid screen. I have shown that WNK1 kinase activity increases in response to osmotic stress, which may imply its kinase activity is important for regulating ion homeostasis in response to a change in cell volume. I have also shown that a proline-rich region of WNK1 interacts with vacuolar protein sorting 4a (VPS4a), an ATPase that helps sort cargo from the plasma membrane to lysosomes. Cells expressing a VPS4 ATP-hydrolysis mutant trap cargo from the cell surface in an aberrant endosomal structure, slowing protein degradation via the lysosomal pathway. I hypothesize that WNK1 delivers cargo to VPS4a to facilitate the degradation of plasma membrane proteins.Item A WNK and a Nudge towards Kinase Biology(2013-01-17) Sengupta, Samarpita; Cobb, Melanie H., Ph.D.A family of four atypical protein kinases; WNKs are characterized by a non-canonical position of the catalytic lysine. The significance of WNKs was first realized when they were found to be causatively linked to a rare form of genetic hypertensive disease known as PHAII. WNKs are an ancient family of kinases and are known to have roles in regulating salt homeostasis in the body in response to osmotic stress, regulating vesicular transport and regulating circadian rhythm in plants. All four WNKs can activate their downstream substrate, OSR1 which in turn can activate ion cotransporters downstream such as NCC and NKCC which results in regulation of ion balance in the cell. WNKs can bind to each other and can potentially form an autoactivable complex. Thus, regulation by WNKs is a complex affair. Further, OSR1 binds to its upstream regulators and substrates via RFxV motifs. WNK1, depending on the splice form, possesses at least five RFxV motifs. I have defined a minimal region on WNK1 required for interaction with OSR1. I have shown that expression of this minimal binding region in cells is sufficient to inhibit OSR1 activation by WNKs. I have also determined that the WNK-OSR1 pathway can cross-talk with the mTORC2 pathway. mTORC2 can directly phosphorylate OSR1 and regulate its activity. Finally, to understand why WNKs possess a slow substrate turnover rate and whether a crucial cofactor is missing, I have determined that WNKs can bind to lipids in vitro. Lipids can alter kinase activity of WNK1 towards OSR1. Future studies will be aimed at understanding the mechanism of action of mTORC2 in regulating the WNK-OSR1 pathway and to determine whether WNKs can bind to lipids in cells and the importance of the lipid binding activity of WNKs. Understanding the intricacies of WNKs would give us important tools to determine its roles in human diseases.Item WNK Family Kinases And The Regulation Of Ion Flux(2007-05-22) Heise, Charles John; Orth, KimThe four mammalian examples of the with no K (lysine) or WNK family of serine/threonine protein kinases have an unusual arrangement of their catalytic lysines. WNK1 and WNK4 have been genetically linked to a rare type of hypertension. Studies demonstrate that these kinases regulate the activity of a diverse group of proteins that in toto suggest WNKs are involved in ionic homeostasis by means of constitutively regulated endocytosis of ion transporters and channels. The mechanism is reliant on an amino terminal fragment of WNK that does not include the kinase domain. How WNK1 exerts this effect, however, remains unclear. This dissertation will detail the examination of the amino terminal fragment of WNK1, its interaction with predicted molecular adaptor proteins, and how these interactions may modulate the activation of the serum- and glucocorticoid-inducible kinase 1, SGK1, leading to the internalization of the epithelial Na+ channel, ENaC via the ubiquitin ligase neuronal precursor cell- expressed, developmentally downregulated protein 4-2, or Nedd4-2.