Browsing by Subject "Phosphoprotein Phosphatases"
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Item CDC14 Coordinates Cyclin Destruction with the Onset of Cytokinesis(2004-08-19) Bembenek, Joshua Nathaniel; Yu, HongtaoThe Cdc14 family of protein phosphatases operate during the final stages of mitosis in various organisms. The Cdc14 phosphatases are downstream components of two homologous signaling pathways: the mitotic exit network (MEN) of S. cerevisiae and septation initiation network (SIN) of S. pombe. Studies of these pathways have revealed divergent roles of Cdc14. In the MEN pathway, Cdc14 is required for cyclin degradation by dephosphorylating Cdh1. The dephosphorylated form of Cdh1 binds to and activates a ubiquitin ligase known as the anaphase-promoting complex (APC/C), which then ubiquitinates mitotic cyclins, targeting them for degradation by the 26S proteosome. In contrast, Cdc14 of the SIN is dispensable for cyclin degradation, but plays an important role during cytokinesis. Two Cdc14 homologues are found in vertebrates, hCdc14A and hCdc14B. I have investigated the regulation of Cdc14 phosphatases to obtain insights into the mechanisms of mitotic exit in higher eukaryotes. Biochemical studies demonstrate that recombinant hCdc14A and hCdc14B can dephosphorylate human Cdh1 and stimulate APC/CCdh1 ligase activity in vitro. Since both the MEN and SIN pathways control Cdc14 localization, I have examined the regulation of the subcellular localization of hCdc14A, hCdc14B and the budding yeast Cdc14. In HeLa cells, hCdc14A localizes to the centrosome whereas hCdc14B is nucleolar during interphase. Both hCdc14 homologues localize to the centrosome and midbody during mitosis. In budding yeast, Cdc14p localizes to the nucleolus during most of the cell cycle and is released in late anaphase when it localizes to the centrosome and the bud neck. The subcellular localization the Cdc14 homologues in HeLa cells is regulated by a nuclear export signal. S. cerevisiae strains carrying only NES mutant CDC14 alleles are capable of degrading mitotic cyclins and escaping mitosis. However, they exhibit a temperature-sensitive phenotype at 37°C because they fail to complete cytokinesis and lack centrosome and bud neck localization of Cdc14. This demonstrates that the Cdc14 phosphatases are regulated by nucleocytoplasmic shuttling. Collectively, my work strongly suggests that the Cdc14 phosphatases play a conserved role in coordinating the destruction of mitotic cyclins with the execution of cytokinesis.Item Characterization of the Role of the PP2A-AB Gene, a Putative Tumor Suppressor, in Cell Growth and Tumorigenesis(2005-05-11) Esplin, Edward D.; Mumby, Marc C.The PP2A-Aβ gene (PPP2R1B) encodes the β isoform of the A subunit of serine/threonine protein phosphatase 2A. Mutations in PP2A-Aβ have been identified in a wide variety of human cancers. The important role of protein phosphatase 2A in down regulating cell growth suggests these mutations may contribute to cancer susceptibility and tumorigenesis by compromising the function of PP2A-Aβ and that PP2A-Aβ may act as a tumor suppressor. Screening of cancer patient DNAs revealed an association between a germline alteration of the PP2A-Aβ and breast carcinoma and identified alterations of PP2A-Aβ in lung carcinoma and ALL patient genomic DNAs. The biochemical consequences of these PP2A-Aβ mutations on PP2A function were investigated by in vitro and in vivo coimmunoprecipitations between the PP2A-Aβ subunit and the B and C subunits of PP2A. These studies showed mutations in PP2A-Aβ confer a loss of function by reducing its ability to bind the B and C subunits, destabilizing the PP2A-Aβ containing PP2A complex. The affect of the PP2A-Aβ gene on cell growth was analyzed by transfecting the PP2A-Aβ gene into cancer cell line deficient for wild type PP2A-Aβ and deriving stable cell lines. The PP2A-Aβ gene appeared to confer a relative disadvantage to transfected cells, resulting in a lower fraction of derived stable lines compared to controls. These cell lines were tested for proliferation and colony formation in soft agar. No significant difference was observed in the growth rate of PP2A-Aβ cell lines compared to controls. One of the PP2A-Aβ stable cell lines demonstrated dramatic suppression of colony formation in soft agar, but this was not confirmed in any additional PP2A-Aβ stable cell lines, leaving this finding inconclusive. The stable cell lines were also analyzed by Western blotting for changes in the Wnt signaling cascade. Cell lines expressing exogenous PP2A-Aβ are found to have lower levels of β-catenin compared to control cell lines. This suggests that the PP2A-Aβ gene is involved in regulating the Wnt signaling pathway, which is shown to be involved in cell growth control and is similarly affected by known tumor suppressor genes.Item Enzymatic Disassembly of Promoter Bound 7SK snRNP Drives Transcription Elongation of HIV and Cellular Genes(2015-11-19) McNamara, Ryan Philip; Cobb, Melanie H.; D'Orso, Iván; Conrad, Nicholas; Kahn, JeffreyGene expression of the human immunodeficiency virus (HIV) and cellular primary responsive genes (PRG’s) is regulated at the step of transcription elongation. Shortly after transcription initiation, RNA Polymerase II (Pol II) pauses and it only enters into productive elongation after inducible transcription factors (TF’s) recruit the P-TEFb kinase to phosphorylate Pol II in response to stimuli. To ensure tight regulation of this process, the majority of P-TEFb is held in a catalytically inactive form, reversibly bound to the 7SK small nuclear ribonucleoprotein (snRNP). In the absence of stimuli, the 7SK snRNP resides in both the nucleoplasm and promoter regions. However, an understanding of how TF’s capture P-TEFb from the 7SK snRNP at the promoter and the mechanism and purpose of localizing the 7SK snRNP to promoters has been largely unexplored. It was therefore my goal to biochemically and functionally characterize this pathway through the use of both the HIV encoded TF Tat and cellular TF’s such as nuclear factor kappa b (NF-κB). Detailed throughout this dissertation, I present the novel findings that HIV Tat and NF-κB function to recruit the PPM1G phosphatase to their targeted promoters, which dephosphorylates P-TEFb and triggers its release from the 7SK snRNP. Additionally, this extraction of P-TEFb from the 7SK snRNP occurs at promoters through the transcriptional regulator KAP1, which physically tethers the snRNP to promoters genome wide. Recruitment of the 7SK snRNP complex occurs after transcription initiation, allowing P-TEFb to be directly positioned for rapid extraction by TF’s upon stimuli and transferred onto the paused Pol II. The enzymatic uncoupling of P-TEFb from the promoter bound 7SK snRNP enables rapid Pol II elongation and gene expression in response to stimuli (for PRG’s) or in the presence of Tat (for HIV). Ultimately, these findings indicate that inducible transcription programs can rapidly respond to environmental cues through the localized positioning of elongation factors at promoters. Moreover, these findings illustrate that HIV has evolved to hijack a cellular gene expression program, thus leading to viral takeover of the host and progression of AIDS.Item Regulation of Hepatic Glycogen Metabolism by Glycogen Targeting Subunits of Protein Phosphatase 1(2007-05-01) Clark, Catherine Renee; Newgard, Christopher B.Glycogen targeting subunits of protein phosphatase 1 play a critical role in fuel homeostasis through the regulation of glycogen metabolism. Adenovirus-mediated overexpression of the liver subunit GL, the muscle subunit GM, or a truncated version of GM, GMdeltaC increased hepatic glycogen content in high fat fed rats, a model of insulin resistance. Rats expressing GMdeltaC and GL had similar amounts of hepatic glycogen following an oral glucose tolerance test, but only GMdeltaC expression improved glucose tolerance. The explanation for this difference is that animals with overexpressed GMdeltaC experience a larger increment in hepatic glycogen storage during OGTT than animals with overexpressed GL, probably related to the much higher fasting liver glycogen levels in the latter group. Since it is possible to improve glucose tolerance via expression of glycogen targeting subunits, the remaining research focused on designing and testing a dominant-negative glycogen targeting subunit, PTG-VF. Overexpression of PTG-VF caused an 83% reduction in glycogen content in hepatocytes indicating that the activity of glycogen targeting subunits is necessary for glycogen accumulation. Further studies found that PTG-VF was more potent in blocking glycogen synthesis in hepatocytes with overexpressed GL than PTG. PTG-VF expression increases phosphoryalse a levels, which preferentially inhibits GL through its C-terminal phosphorylase a binding site that is lacking in PTG. Removal of the phosphorylase a binding site from GL renders the subunit less susceptible to inhibition by PTG-VF. PTG-VF was overexpressed in rats fed on standard chow (SC) or high fat (HF) diet to determine if suppression of glycogen targeting subunit activity could cause glucose intolerance or diabetes. Hepatic glycogen stores were decreased by PTG-VF, but this did not lead to alterations in glucose homeostasis.