Biochemistry Commons^™

Proteomic Approaches To Identify Unique And Shared Substrates Among Kinase Family Members, Charles Lincoln Howarth

Dartmouth College Ph.D Dissertations

Protein phosphorylation is a reversible post-translational modification that is a critical component of almost all signaling pathways. Kinases regulate substrate proteins through phosphorylation, and nearly all proteins are phosphorylated to some extent. Crucially, breakdown in phosphorylation signaling is an underlying factor in many diseases, including cancer. Understanding how phosphorylation signaling mediates cellular pathways is crucial for understanding cell biology and human disease.

Targeted protein degradation (TPD) is a strategy to rapidly deplete a protein of interest (POI) and is applicable to any gene that is amenable to CRISPR-Cas9 editing. One TPD approach is the auxin-inducible degron (AID) system, which relies …

Chemically Diverse Microtubule Stabilizing Agents Initiate Distinct Mitotic Defects And Dysregulated Expression Of Key Mitotic Kinases., Cristina C Rohena, Jiangnan Peng, Tyler A. Johnson, Phillip Crews, Susan L Mooberry

Tyler Johnson

Microtubule stabilizers are some of the most successful drugs used in the treatment of adult solid tumors and yet the molecular events responsible for their antimitotic actions are not well defined. The mitotic events initiated by three structurally and biologically diverse microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel were studied. These microtubule stabilizers cause the formation of aberrant, but structurally distinct mitotic spindles leading to the hypothesis that they differentially affect mitotic signaling. Each microtubule stabilizer initiated different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ causes centrosome separation and disjunction failure to a much greater extent …

Regulation Of Alteration/Deficiency In Activation 3 (Ada3) By Acetylation And Its Role In Cell Cycle Regulation And Oncogenesis, Shashank Srivastava

Theses & Dissertations

The ADA3 (Alteration/Deficiency in Activation 3) protein is a transcriptional adaptor protein that was initially discovered as a component of several HAT (Histone Acetyltransferase) complexes, the enzyme complex responsible for histone acetylation, which is a prerequisite for transcription. Earlier the studies from Dr. Band’s laboratory and that of others’ have deciphered a crucial role of ADA3 in cell cycle regulation (both through G₁/S and G₂/M phase transitions) and in maintaining the genomic stability.

While our laboratory investigated the mechanism behind the role of ADA3 in G₁/S transition, the same remained unknown for G_{2 …}

Biochemical And Structural Characterization Of Cdc14 Phosphatases From Pathogenic Fungi, John Whitney, Kyle Wettschurack, Mark Hall, Jeremy Lohman, Aaron Benjamin, Lee M. Stunkard

The Summer Undergraduate Research Fellowship (SURF) Symposium

Cyclin-dependent kinases (Cdk) drive cell cycle progression and reversal of Cdk phosphorylation is essential for mitotic exit. Cdc14 is a widely conserved family of protein phosphatases that reverse Cdk phosphorylation. Recently, Cdc14 was also found to be essential for pathogenicity of some fungal plant pathogens. Fungal pathogens, like Ustilago maydis, decrease agricultural crop yield costing global agriculture by some accounts $60 billion per year. Since Cdc14 is absent in plants, a fungi specific Cdc14 inhibitor could be made to reduce the pathogenicity of U. maydis and other fungal plant pathogens to increase crop yields. To guide inhibitor development, a …

Characterization And Target Identification Of Ak301: A Novel Mitotic Arrest Agent, Michael J. Bond, Avijeet S. Chopra, Marina Bleiler, Michelle Yeagley, Eric Scocchera

University Scholar Projects

The Giardina Laboratory has recently identified AK301 as a novel mitotic arrest agent. This work aimed to characterize the arrest state induced by AK301 (EC₅₀ ~ 150nM) and identify the cellar targets responsible for the arrest. It was found that AK301 arrest is readily reversible upon withdrawal of AK301. Cells that slip from mitosis after removal of AK301 are sensitized to apoptosis. This was found to be unique for AK301 when compared to other mitotic arrest agents like colchicine, vincristine, and BI2536. Arrested cells were found to have increased ATM activity as well as an upregulation of p53 and …

Interaction Between Atm Kinase And P53 In Determining Glioma Radiosensitivity, Syed F. Ahmad

Theses and Dissertations

Glioblastoma multiforme (GBM) is the most common primary brain tumor. Studies have shown that targeting the DNA damage response can sensitize cancer cells to DNA damaging agents. Ataxia telangiectasia mutated (ATM) is involved in signaling DNA double strand breaks. Our group has previously shown that ATM inhibitors (ATMi) sensitize GBM cells and tumors to ionizing radiation. This effect is greater when the tumor suppressor p53 is mutated.

The goals of this work include validation of a new ATM inhibitor, AZ32, and elucidation of how ATMi and p53 status interact to promote cell death after radiation. We propose that ATMi and …

Chemically Diverse Microtubule Stabilizing Agents Initiate Distinct Mitotic Defects And Dysregulated Expression Of Key Mitotic Kinases., Cristina C. Rohena, Jiangnan Peng, Tyler A. Johnson, Phillip Crews, Susan L. Mooberry

Natural Sciences and Mathematics | Faculty Scholarship

Microtubule stabilizers are some of the most successful drugs used in the treatment of adult solid tumors and yet the molecular events responsible for their antimitotic actions are not well defined. The mitotic events initiated by three structurally and biologically diverse microtubule stabilizers; taccalonolide AJ, laulimalide/fijianolide B and paclitaxel were studied. These microtubule stabilizers cause the formation of aberrant, but structurally distinct mitotic spindles leading to the hypothesis that they differentially affect mitotic signaling. Each microtubule stabilizer initiated different patterns of expression of key mitotic signaling proteins. Taccalonolide AJ causes centrosome separation and disjunction failure to a much greater extent …

Cdk1 And Plk1 Mediate A Clasp2 Phospho-Switch That Stabilizes Kinetochore–Microtubule Attachments, Ana R. R. Maia, Zaira Garcia, Lilian Kabeche, Marin Barisic

Dartmouth Scholarship

Accurate chromosome segregation during mitosis relies on a dynamic kinetochore (KT)-microtubule (MT) interface that switches from a labile to a stable condition in response to correct MT attachments. This transition is essential to satisfy the spindle-assembly checkpoint (SAC) and couple MT-generated force with chromosome movements, but the underlying regulatory mechanism remains unclear. In this study, we show that during mitosis the MT- and KT-associated protein CLASP2 is progressively and distinctively phosphorylated by Cdk1 and Plk1 kinases, concomitant with the establishment of KT-MT attachments. CLASP2 S1234 was phosphorylated by Cdk1, which primed CLASP2 for association with Plk1. Plk1 recruitment to KTs …

Mitotic Regulation Of Protein Kinase Ck2, Nicole A. St. Denis

Electronic Thesis and Dissertation Repository

Protein kinase CK2 is a serine/threonine kinase with a multitude of substrates and roles in many cellular processes, including mitosis. CK2 is constitutively active, yet we hypothesize that CK2 is indeed regulated in mitosis through subtle means, enabling CK2 to perform its functions unique to cell division. Our aims were to examine the roles of mitotic phosphorylation, subcellular localization, and interplay with mitotic kinases in the regulation of CK2 activity.

We first examined the role of four highly conserved mitotic phosphorylation sites located in the unique C-terminus of CK2α. Phosphospecific antibodies generated against the sites show that CK2α phosphorylation is …

The Kini Kinesin Kif2a Is Required For Bipolar Spindle Assembly Through A Functional Relationship With Mcak, Neil J. Ganem, Duane A. Compton

Dartmouth Scholarship

Although the microtubule-depolymerizing KinI motor Kif2a is abundantly expressed in neuronal cells, we now show it localizes to centrosomes and spindle poles during mitosis in cultured cells. RNAi-induced knockdown of Kif2a expression inhibited cell cycle progression because cells assembled monopolar spindles. Bipolar spindle assembly was restored in cells lacking Kif2a by treatments that altered microtubule assembly (nocodazole), eliminated kinetochore–microtubule attachment (loss of Nuf2), or stabilized microtubule plus ends at kinetochores (loss of MCAK). Thus, two KinI motors, MCAK and Kif2a, play distinct roles in mitosis, and MCAK activity at kinetochores must be balanced by Kif2a activity at poles for spindle …

Minus-End Capture Of Preformed Kinetochore Fibers Contributes To Spindle Morphogenesis, Alexey Khodjakov, Lily Copenagle, Michael B. Gordon, Duane A. Compton, Tarun M. Kapoor

Dartmouth Scholarship

Near-simultaneous three-dimensional fluorescence/differential interference contrast microscopy was used to follow the behavior of microtubules and chromosomes in living alpha-tubulin/GFP-expressing cells after inhibition of the mitotic kinesin Eg5 with monastrol. Kinetochore fibers (K-fibers) were frequently observed forming in association with chromosomes both during monastrol treatment and after monastrol removal. Surprisingly, these K-fibers were oriented away from, and not directly connected to, centrosomes and incorporated into the spindle by the sliding of their distal ends toward centrosomes via a NuMA-dependent mechanism. Similar preformed K-fibers were also observed during spindle formation in untreated cells. In addition, upon monastrol removal, centrosomes established a transient …

Numa Assembles Into An Extensive Filamentous Structure When Expressed In The Cell Cytoplasm, Alejandro Saredi, Louisa Howard, Duane A. Compton

Dartmouth Scholarship

NuMA is a 236 kDa protein that participates in the organization of the mitotic spindle despite its strict localization in the nucleus during interphase. To test how cells progress through mitosis when NuMA is localized in the cytoplasm instead of the nucleus, we have deleted the nuclear localization sequence of NuMA using site-directed mutagenesis and transiently expressed this mutant protein (NuMA-DeltaNLS) in BHK-21 cells. During interphase, NuMA-DeltaNLS accumulates in the cytoplasm as a large mass approximately the same size as the cell nucleus. When cells enter mitosis, NuMA-DeltaNLS associates normally with the mitotic spindle without causing any apparent deleterious effects …