Browsing by Subject "Protein-Serine-Threonine Kinases"

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Analysis of Aurora B Regulation and Signaling
(2006-05-16) Öncel, Dilhan; Yu, Hongtao
Aurora 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.
Autoinhibition and Chloride Sensing in the WNK1 Kinase
(2012-07-16) Moon, Thomas Matthew; Goldsmith, Elizabeth J.
Protein 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.
Characterizing New Molecules and Mechanisms of Semaphorin/Plexin/MICAL Signaling
(2013-07-16) Yoon, Jimok; Krämer, Helmut; Cobb, Melanie H.; Henkemeyer, Mark; Terman, Jonathan R.
The mechanisms that regulate the cellular behaviors including morphology, motility, navigation, and connectivity that are critical for normal human health are still incompletely understood. These types of behaviors are regulated through the ability of guidance cues that are present outside of cells to exert precise effects on the cell’s internal actin cytoskeleton. To help characterize an underlying logic for these cellular changes and proper cellular function I have been characterizing how one of the largest families of extracellular guidance cues, members of the Semaphorin family of proteins, induces actin cytoskeletal changes. Interestingly, Semaphorins have been found to inhibit the movement of cells and their membranous processes but the molecules linking them to these specific behaviors have remained poorly understood. Therefore, during my graduate work, I began further characterizing a new family of proteins, the MICALs that were identified as cytoplasmic binding partners of the Semaphorin receptor Plexin. Combining Drosophila genetics with in vitro biochemical assays, my work revealed that Mical regulates actin organization both in vivo and in vitro and is a novel actin disassembly factor. These results provide a new basis for understanding how extracellular guidance cues regulate the actin cytoskeleton. I then went on to further explore Mical-mediated actin filament (F-actin) disassembly. In one line of investigation, my work revealed that Mical plays an antagonistic role to F-actin stabilizing/bundling proteins including fascin and espin in regulating the F-actin cytoskeleton in vivo. This work also indicated that Semaphorin/Plexin/Mical activity not only directly disassembles the F-actin cytoskeleton but also triggers other actin regulatory proteins to reorganize a more complex F-actin network, resulting in increased cellular plasticity. In another line of investigation, I found that the Abl non-receptor tyrosine kinase is a new Mical-interacting protein. My functional assays revealed that Abl and Semaphorin/Plexin/Mical work together to regulate F-actin arrangements and these interactions are conserved in many different contexts including bristle cell morphology, axon guidance, and cancer cell survival and invasion. Thus, I have found that MICAL family proteins are novel controllers of the actin cytoskeleton, functioning directly on F-actin and with other actin regulatory proteins to modulate diverse cellular behaviors.
Context-Selective Support of the AKT/mTOR Regulatory Axis by Tank-Binding Kinase 1 (TBK1)
(2016-11-28) Cooper, Jonathan Mark; Brugarolas, James B.; Cobb, Melanie H.; Brekken, Rolf A.; White, Michael A.
Oncogenic mutation of Ras or Ras effector signaling characterizes roughly thirty percent of all cancers. Persistent obstacles to the treatment of these diseases by direct Ras inhibition prompt alternative strategies aimed at leveraging signaling networks downstream of Ras. Tank-Binding Kinase 1 (TBK1) is downstream of the RalGEF/RalB arm of Ras effector signaling and supports Ras-driven oncogenic transformation via direct regulation of AKT. While TBK1 has been nominated as a therapeutic target, the field lacks knowledge of the mechanisms whereby TBK1 inhibitors mediate lethality and of the preferential context(s) for their application. We therefore leveraged toxicity profiles for TBK1 inhibitors in 100 NSCLC cell lines and identified robust correlation between TBK1 inhibitors and a cadre of mTOR direct and upstream regulatory network signaling inhibitors. This observation, along with orthogonal phosphoproteomics data, suggested an intersection exists between TBK1 and mTOR regulation and mechanistic target space. We identified that TBK1 is required for AKT/mTOR activation during the nutrient starved-to-fed state transition. Furthermore, we established that TBK1 physically intersects with the AKT/mTOR regulatory axis signaling at multiple nodes and can follow permissive and instructive mechanistic routes to regulate mTORC1 activation in response to nutrients. In parallel, we utilized a bioinformatics approach to identify that "Ras-mutant/mesenchymal" status serves as a molecular indicator of TBK1 inhibitor sensitivity in NSCLC. Concordantly, signaling through the AKT/mTOR regulatory axis was acutely attenuated by TBK1 inhibition in Ras-mutant/mesenchymal but remained unresponsive in Ras-mutant/epithelial NSCLC, indicating TBK1-resistant NSCLC may have uncoupled AKT/mTOR signaling from substantive TBK1 regulation. We furthermore demonstrated that TBK1 inhibition synergizes with Transforming Growth Factor-beta (TGF-beta)-mediated induction of the epithelial-to-mesenchymal transition (EMT) to reduce cancer cell viability. Together, these observations suggest that TBK1 supports pro-survival signaling downstream of Ras and EMT/TGF-beta signaling through the AKT/mTOR regulatory axis. Our findings, therefore, reveal novel mechanistic contributions of TBK1 in the regulation of AKT/mTOR signaling, and also nominate Ras-mutant/mesenchymal NSCLC as the preferential context for therapeutic interventions targeting TBK1.
Dissection of Mechanisms Regulating the Drosophila Hedgehog Pathway
(2012-07-20) Shi, Qing; Jiang, Jin
Hedgehog (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.
Dynamic Modulation of Exocyst Interaction Networks Integrates Hippo and mTOR Pathway Response to Pathogen Detection
(2017-11-16) Zaman, Aubhishek; Brekken, Rolf A.; White, Michael A.; Schmid, Sandra; Mirzaei, Hamid
Monomeric RALGTPases, via direct binding to the exocyst (a.k.a Sec6/8 complex), help mount productive cell autonomous responses to trophic and immunogenic signals. However, RAL-exocyst downstream effectors register in a bewildering array of signaling events-- suggesting the presence of mechanisms that confer context dependent coordination amongst them. Here, we employed quantitative proteomics-based characterization of dynamic signal-dependent modulation of the exocyst interactome as an approach to detect such mechanisms. We identified sentinel innate immune kinases- PKR and TBK1 as host defense stimulus induced effectors of distinct exocyst subcomplexes specified by the presence of Exo84 versus Sec5 subunits. We find that, under virally compromised conditions, the Exo84 subcomplex accommodates Hippo signaling kinase MST1 together with PKR whereas a Sec5 subcomplex assists assembly of anabolic growth regulatory kinase mTOR together with TBK1. Detailed functional and biochemical analysis indicated that PKR directly phosphorylates MST1 for activation of Hippo signaling and consequent YAP1 inactivation. In parallel, TBK1 was found to be a positive regulator for mTOR and a negative regulator of YAP1 activity. Furthermore, RALB, which is activated by the host defense response, was found to be required and sufficient for induction of both Hippo and mTOR signaling through dual exocyst subcomplex engagement. RALB-dependent activation of these pathways can help cells deflect viral challenge and can be corrupted by oncogenes to help deflect apoptotic checkpoint activation. Thus, RAL-exocyst signaling complexes can be recognized as context-dependent mechanisms for integrated engagement of Hippo and mTOR signaling pathways.
Evaluation of Chronic RalGTPase Activation as a Core Specifier of Oncogenic Transformation
(2009-01-08) Cheng, Tzuling; White, Michael A.
Ral (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.
Functional Genomics Based Interrogation of Cell-Fate Determination Pathways
(2011-08-10) Jacob, Leni Susan; Lum, Lawrence
The 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.
The Hippo Signaling Pathway in Organ Size Control and Regeneraton
(2012-07-17) Ren, Fangfang; Jiang, Jin
The 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.
Identification of Substrates and Pathways Regulated by PAS Kinase
(2005-12-20) Probst, Brandon Linn; McKnight, Steven L.
PAS 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.
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.
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.
Investigating the Biological Functions of the Protein Kinase WNK1 in the Regulation of Cytoskeletal Structures and Membrane Trafficking
(2013-06-19) Tu, Szu-Wei; Luby-Phelps, Katherine; Cobb, Melanie H.; Albanesi, Joseph P.; Rice, Luke M.
With No Lysine (WNK) 1, a serine/threonine kinase, is a unique kinase to its catalytic lysine residue at a non-canonical position relative to all other kinases. Characterization of endogenous WNK1 distribution by immunofluorescence reveals a perinuclear punctate pattern. I have investigated this perinuclear distribution and how it might relate to the biological functions of WNK1 from two aspects. First, I investigated cytoskeletal structures mainly focused on the microtubules. WNK1 localized on mitotic spindles during mitosis as well as interphase microtubules. Depletion of WNK1 caused aberrant mitotic spindles, chromosomes and defect of abscission. In interphase cells, disruption of radiating microtubules from microtubule organization center was observed. Centrosomal structure was impaired. Cells showed a migratory defect. Clues from a former student and my mass spectrometry data suggested that dynein and its associated protein-dynactin, centrosomal protein of 70 and 170 kDa might be potential interactors mediating microtubule related phenotypes. Second, I examined WNK1 and membrane trafficking events. Depletion of WNK1 caused higher amount of epidermal growth factor receptors remained at the later step of endocytosis. Lysosomes and lysosome-related organelles were disrupted. Biochemical assay suggested that WNK1 could associate with active Rab 6 or 7 effector complexes. I have identified that the homotypic fusion and vacuole protein sorting (HOPS) complex, one of Rab7 effector complexes could interact with WNK1. Mass spectrometry results showed that WNK1 could pull down clathrin heavy chain and adaptor protein complex-3 (AP-3) β subunit. AP-3 vesicles are also HOPS complex-mediated vesicular trafficking between the Trans-Golgi network and late endosomes. Co-localization analysis suggested that WNK1 co-localized with AP-3 in a high pearson correlation coefficient (0.53). Depletion of WNK1 showed defect of the maturation of autophagosomes. Taken together, WNK1 might affect membrane trafficking through HOPS complex-mediated homotypic (the assembly of phagophores) and heterotypic (late endosomes and lysosomes) membrane fusion.
LKB1, CCL2, and Macrophages: A New Axis of Endometrial Cancer Progression
(2015-04-07) Peña, Christopher George; Brekken, Rolf A.; Lum, Lawrence; Amatruda, James F.; Castrillon, Diego H.
Cancer of the uterus is a common malignancy in women with no adequate treatments for tumors that have progressed beyond the uterus. The serine-threonine kinase LKB1 has been identified as a potent suppressor of uterine cancer. Combined genetic, proteomic, and in vivo studies in genetically engineered mouse models show that loss of LKB1 protein is associated with high grade, high stage tumors with unfavorable clinical outcomes. However, the mechanism(s) by which LKB1 drives malignant transformation of uterine cancers remains unclear. Here I show that LKB1 unexpectedly suppresses tumor progression via pAMPK dependent secretion of the inflammatory cytokine CCL2. Lkb1 inactivation in vivo resulted in abnormal production of CCL2, which led to recruitment of pro-tumorigenic macrophages (aka immunosuppressive macrophages) responsible for tumor invasion. Conditional inactivation of Ccl2 in an Lkb1-driven mouse model of endometrial cancer slowed tumor progression, increased survival, and significantly reduced infiltration of macrophages in the tumor microenvironment. In human primary endometrial cancers (EMCAs), loss of LKB1 protein was strongly associated with increased CCL2 and macrophage density. Additionally, high stage and high grade EMCAs were characterized by loss of LKB1 protein, elevated production of CCL2, and increased macrophage density. These data demonstrate that CCL2 is a potent effector of LKB1 loss in endometrial cancer, creating new therapeutic opportunities for targeting CCL2 and the tumor microenvironment.
Loss of TBK1 Kinase Function Improves Disease Outcome in Pancreatic Cancer and Metabolic Syndrome
(2018-07-30) Cruz, Victoria Haley; Castrillon, Diego H.; Brekken, Rolf A.; MacDonald, Raymond J.; Scherer, Philipp
Aberrant expression and activity of TANK binding kinase 1 (TBK1) has been observed in numerous diseases. Here I've identified novel functions for TBK1 in pancreatic ductal adenocarcinoma (PDA) and in metabolic syndrome that promote disease progression. Activating mutations in KRAS are present in 90% of human PDA cases; yet direct pharmacological inhibition of K-RAS remains a challenge, indicating a need for effective therapies. Higher levels of TBK1 mRNA, a critical downstream mediator of oncogenic K-RAS in lung cancer, correlate with poorer outcome in PDA patients. Given these observations, I hypothesized that TBK1 is also an effector of K-RAS in PDA. KRAS mutant PDA cell lines are selectively sensitive to small molecule inhibition of TBK1. In K-RAS-driven genetic mouse models of PDA, Tbk1 supports spontaneous pancreatic tumor growth as evidenced by smaller tumors and fewer metastases in Tbk1 mutant PDA mice relative to normal PDA mice. Additionally, Tbk1 mutant tumors are more epithelial; an observation consistent with the reduced migratory phenotype of Tbk1 mutant tumor cell lines and lack of detectable metastases in Tbk1 mutant PDA animals. Mechanistic studies indicate that TBK1 is central to Axl-driven EMT and is activated with RAS in response to Axl stimulation in PDA cell lines. The latter part of this thesis is focused on the contribution of TBK1 to mice with metabolic disorder. TBK1 is implicated in the regulation of metabolism through studies with amlexanox, an inhibitor of IκB kinase (IKK)-related kinases. Amlexanox induced weight loss, reduced fatty liver and insulin resistance in high fat diet (HFD) fed mice and has now progressed into clinical testing for the treatment and prevention of obesity and type 2 diabetes. However, since amlexanox is a dual IKKε/TBK1 inhibitor, the specific contribution of TBK1 is unclear. To distinguish metabolic functions unique to TBK1, I examined the metabolic profile of global Tbk1 mutant mice challenged with HFD and investigated potential mechanisms for the improved metabolic phenotype. I report that systemic loss of TBK1 kinase function has a protective effect on metabolic readouts in HFD-fed mice, which is mediated by loss of an inhibitory interaction between TBK1 and the insulin receptor.
Mechanistic Studies of Autophagy Initiation in Mammalian Cells
(2011-08-10) Shang, Libin; Wang, Xiaodong
Macroautophagy (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.
Molecular Mechanisms Underlying Innate Immune Kinase TBK1-Driven Oncogenic Transformation
(2013-04-16) Ou, Yi-Hung 1977-; Lum, Lawrence; White, Michael A.; Cobb, Melanie H.; Minna, John D.
An essential kinase in innate immune signaling, TBK1 couples pathogen surveillance to induction of host defense mechanisms. The pathological activation of TBK1 in cancer can overcome programmed cell death cues, enabling cells to survive oncogenic stress. The mechanistic basis of TBK1 prosurvival signaling, however, has been enigmatic. Here we show that TBK1 directly activates AKT by phosphorylation of the canonical activation loop and hydrophobic motif sites independently of PDK1 and mTORC2. A population of AKT is bound to components of the exocyst complex. Upon mitogen stimulation, triggering of the innate immune response, re-exposure to glucose, or oncogene activation, TBK1 is recruited to the exocyst, where it activates AKT. In cells lacking TBK1, insulin activates AKT normally, but AKT activation by these exocyst-dependent mechanisms is impaired. Discovery and characterization of a 6-aminopyrazolopyrimidine derivative, as a selective low nanomolar TBK1 inhibitor, indicates this regulatory arm can be pharmacologically perturbed independently of canonical PI3K/PDK1 signaling. Thus, AKT is a direct TBK1 substrate that connects TBK1 to prosurvival signaling. Additionally, biochemical and cell biological evidence indicates critical roles of TBK1 and its analog IKKε in the amino acid-dependent activation of mTORC1. TBK1 and IKKε are activated by amino acids and both proteins interact with mTORC1. In TBK1 and/or IKKε-deficient cells, mTORC1 activation by amino acids is impaired. Of note, we also discovered a set of TBK1 substrates and interacting proteins participating in amino acid-dependent mTORC1 signaling. In conclusion, our results suggest that TBK1 not only supports physiological and oncogenic activation of AKT, but also plays a central role in the regulation of mTORC1 activation in response to amino acids. In addition, our studies reveal novel mTORC1 components and provide new insights into the regulation of the mTORC1 signaling network.
The Multifunctional Kinase Bub1 Acts as a Signaling Hub for the Spindle Checkpoint
(2015-11-12) Jia, Luying; De Martino, George; Yu, Hongtao; Cobb, Melanie H.; White, Michael A.
The spindle checkpoint is an essential mechanism to ensure accurate chromosome segregation during mitosis. The checkpoint signal originates from the kinetochore, which is a huge protein assembly on centromeric chromatin. Kinetochore is also the receptor for spindle microtubules, which enables it to translate microtubule attachment status into spindle checkpoint signal. The separation of the sister chromatids and the progression from metaphase to anaphase requires the activation of an ubiquitin E3 ligase, anaphase-promoting complex or cyclosome (APC/C). Cdc20 is the mitosis-specific APC/C activator. The spindle checkpoint prevents premature sister chromatids separation by preventing Cdc20 from activating APC/C. Bub1 is a highly conserved spindle checkpoint protein that plays multiple roles in checkpoint signaling. On the kinetochore, Bub1 recruits other important checkpoint proteins like BubR1, Mad1 and Cdc20. We found phosphorylation on Bub1 serine 459 is essential for spindle checkpoint and for Bub1-Mad1 interaction. However, the majority of Mad1 still localize to the kinetochore in cells expressing Bub1-S459A mutant. These results suggest that the direct binding between Bub1 and Mad1 through Bub1-S459 may not be responsible for the localization of Mad1 to the kinetochore region. Instead, this interaction enables Mad1 to function in the checkpoint signaling pathway, possibly through regulating its interaction with Bub1-bound BubR1 and Cdc20. Bub1 is also a serine/threonine kinase. The only two identified substrates are histone H2A and Cdc20. Bub1 phosphorylates histone H2A threonine 120, which is important in recruiting Sgo1 and Aurora B kinase to the kinetochore. Bub1 also phosphorylates Cdc20 serine 153. It was shown in vitro that phosphorylation by Bub1 can inhibit APC/CCdc20. However, mouse embryonic fibroblasts (MEFs) expressing Bub1 kinase dead mutant only display mild checkpoint defect due to abnormal Aurora B localization. In addition, over-expression of Bub1 kinase dead mutant in HeLa cells can rescue the checkpoint defect caused by Bub1 depletion using siRNA. These results challenged the importance of Cdc20 phosphorylation by Bub1 in the spindle checkpoint. Here I show that Bub1 binds another kinase Plk1, forming a kinase complex. Phosphorylation of Cdc20 by Bub1-Plk1 not only inhibits APC/CCdc20 in vitro, but also is required for proper spindle checkpoint function in HeLa cells.
Regulation and Dysregulation via Docking Interactions in WNK and ERK1/2 MAPK Signaling
(2016-12-07) Taylor, Clinton A., IV; Albanesi, Joseph P.; Cobb, Melanie H.; Goldsmith, Elizabeth J.; Westover, Kenneth D.
Protein-protein interactions are essential for nearly every cellular process. Within signaling pathways, such interactions carry out numerous functions such as defining substrate specificity, inhibition of both other interactions and enzyme activity, localizing signaling partners, acting in a switch-like manner depending on post-translational modifications, etc... My work has focused primarily on protein-protein interactions, and in particular protein-peptide docking interactions, within two independent cellular signaling pathways, the WNK, and ERK1/2 MAPK pathways. The WNK pathway is an essential regulator of cellular ionic composition and volume. Initially discovered due to the genetic link between mutations in some of the WNK kinases and an inherited form of hypertension, the pathway is now well defined and known to regulate the activity of multiple SLC12 cation chloride coupled cotransporters. WNK kinases activate SPAK and OSR1 kinases, which then regulate the activity of cotransporters. My initial work within this pathway was focused on understanding the role of SPAK and OSR1 activation loop domain-swapping. I solved the structure of the inactive SPAK kinase domain, and utilized a previously identified dimerization blocking mutation to probe the role of SPAK dimerization on activation and activity. I determined that SPAK has multiple activation states and that the monomeric form is both active and can be activated by WNK1. I next shifted focus to the SPAK and OSR1 CCT domains, which mediate protein-peptide interactions with motifs found in WNKs, cotransporters, and other interaction partners. I further defined the specificity of the CCT domains for the motifs, and discovered a new motif variant. I used this information to predict new interaction partners. Although validation of the predictions is still in the early stages, my initial choice for validation was a group of inward rectifier potassium channels. Through collaboration, we now have evidence that OSR1 kinase activity regulates flux through one of the eight channels, and testing of more of the channels is underway. The ERK1/2 MAPK pathway is involved in numerous cellular processes, and is well known for its role in responding to extracellular signals that regulate cell growth and differentiation. As such, aberrant signaling within the pathway is found within approximately thirty percent of all cancers. The terminal kinases of the cascade, ERK1 and ERK2, phosphorylate hundreds of cytosolic and nuclear substrates, and in many cases protein-peptide docking interactions between ERK1/2 and substrates are required. One of the docking sites on ERK2 has been shown to be the site of a mutation, E322K, that is the most enriched ERK2 missense mutation in human cancers. Using a previously determined dataset, I was able to refine the structure of the mutant ERK2. As expected the site of the mutation was structurally disrupted, but the mutation also caused structural changes throughout the entire kinase domain, including solvent exposure of the activation loop. Docking induced solvent exposure of the activation loop had already been suggested to be important for MAPK activation and inactivation by presenting the sites of phosphorylation to modifying enzymes. Therefore, the structural data fit well with previous findings indicating that the mutant was able to be activated by upstream kinase MEK1, but could not be inactivated by phosphatase DUSP6. I also found that the mutation affected a distal docking site and disrupted interactions with at least some interaction partners at that site. The combined results of this project have shown that this mutation has multiple effects on ERK2 structure and function, and implies that this mutation is enriched primarily because it allows ERK2 to be activated similar to wild type, while having a diminished capacity to be deactivated.
Regulation and Mechanism of Bub1-Mediated Spindle Checkpoint Signaling
(2006-12-20) Qi, Wei; Yu, Hongtao
The 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.