Thesis (M.S.)--Brigham Young University. Dept. of Physiology and Developmental Biology, 2006. / Includes bibliographical references (p. 37-44).

Alzheimer¡¦s disease (AD), one of the most common dementia, is characterized by the formation two types of aggregation in the brain: senile plaques and neurofibrillary tangles (NFTs). NFTs are composed of hyperphosphorylated Tau. Tau protein mainly expressed in brain and was identified as one of the microtubule-associated proteins (MAPs). Hyperphophorylation on Tau affects its binding to tubulin and capacity to promote microtubule assembly. A number of proline-directed kinase capable of phosphorylating PHF-Tau have been identified, including Glycogen Synthase Kinase-3£] (GSK-3£]). Here we demonstrated that GSK3£] can co-purify with PHFs and can co-localize with Tau in vitro in N18 cells. To examine the phosphorylation mechanism of Tau by GSK-3£], N-terminal, C-terminal, T231A, T231E, 154~441, S396A, S400A, S404A, S413A and S396A S400A mutants of Tau were used, respectively. We were able to demonstrate that phosphorylation on Thr231 and Ser404 in Tau may play important roles for GSK3£] phosphorylation and its functional regulation. Most importantly, we have proved that T231P motif is necessary and critical for Tau phosphorylation by GSK3£]. Moreover, we used T231E, S396E and S400E mutants of Tau to understand the functional regulation of Tau by GSK3£] phosphorylation by tubulin assembly assay. Surprisingly, we observed all of these Tau mutants can promote tubulin assembly and form tubulin bundles in N18 cells. It has been proved that Pin1 WW domain can bind to Cdc2-phosphorylated Thr-231-Pro motif of Tau and restore the ability of Tau to promote tubulin assembly. In this study, we also studied whether Pin1 can regulate GSK3£]- phosphorylated Tau. The results show that Pin1 WW domain can bind to phosphorylated Thr-231 of Tau by GSK3£] and restore the ability of Tau to promote tubulin assembly.

Functional inactivation of tumor susceptibility gene tsg101 leads to cellular transformation and tumorigenesis in mice. No genomic DNA deletion in TSG101gene in human cancer indicated TSG101 is not a typical tumor suppressive gene. TSG101 participates in the MDM2/p53 feedback control loop and the regulation of the cellular membrane trafficking. However, detail functional characteristics remains to be elucidate. In this study, we explored the tsg101 expression in adult mouse tissues from various organs using immunohistochemistry and in situ hybrid- ization. The results indicated that tsg101 expression was ubiquitous but in differential steady-state level in various cell types. The expression of tsg101 mainly found in epithelial cells¡Bsecretory cells and nerve cells. The second topic of this study was to characterize the phosphorylation status of TSG101 protein. Endogeneously expressed TSG101 and exogeneously expressed HA-tag TSG101 protein were purified by immunoprecipiation with #820 antiserum against TSG101, and were subjected for western blot analysis using anti-phosphoserine and anti-phosphothreonine antibodies. This experiment had confirmed that TSG101 protein contained both phosphoserine and phosphthreonine residues. In vitro kianse assay using GST-tag and his-tag TSG101 funsion proteins was exploited to investigate the kinase responsible for TSG101 phosphorylation. The results clearly indicated that cdc2¡BGSK3£] and PKC kinases could phosphorylate TSG101 fusion Protein, implying that the function of TSG101 might be regulated by the signaling involving these kinases.

Diss.--Åbo Akademi University, 2003. / Contient cinq articles publiés par l'auteur dans des revues scientifiques. Bibliogr. en fin de chap.

Thesis (Ph. D.)--University of Alberta, 2010. / Title from pdf file main screen (viewed on Jan. 18, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy, Department of Biochemistry, University of Alberta. Includes bibliographical references.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 92-98).

Includes bibliographical references (p. 64-71).

published_or_final_version / Zoology / Master / Master of Philosophy

Oxysterol binding protein (OSBP) binds 25-hydroxycholesterol (25OH) and cholesterol, which regulates PH and FFAT domain interaction with the Golgi apparatus and endoplasmic reticulum, respectively. Adjacent to these domains is a phosphorylated serine-rich motif (SRM, T379, S381, S384, S387, S388, S391) that we hypothesize controls sterol transport by OSBP. To test this, OSBP dephospho-mimics or phospho-mimics were expressed in CHO cells. Western blot analysis showed that the S381 is phosphorylated by PKA and is required for phosphorylation of down-stream serine residues. When expressed in OSBP-null CHO cells, there was no difference in the localization of the OSBP mutants, and all mutants restored SM synthesis in response to 25OH. Recombinant OSBP 5S?5E had increased cholesterol binding and extraction, and decreased cholesterol transfer to liposomes compared to OSBP. OSBP 5S?5E also bound VAP more efficiently. A model is proposed wherein SRM phosphorylation facilitates VAP association with the ER and increases cholesterol extraction.

Using a novel purification scheme and a new assay for detection of DNA kinase activity, a Polymin P-precipitable DNA kinase has been identified and characterized from calf thymus extracts. The DNA kinase activity was able to phosphorylate RNA as well as single-stranded and double-stranded DNA, therefore it has been termed Polymin P-precipitable polynucleotide kinase (PP-PNK). The enzyme had a neutral to alkaline, broad pH optimum that distinguished it from the previously described mammalian DNA kinases that have an acidic pH optimum. The sedimentation coefficient of the enzyme was 3.4-3.8 S, indicating a molecular weight of about 50 kDa. Estimates for the K$ sb{ rm M}$ for ATP were 52 $ mu$M and for the oligonucleotide substrate 8 $ mu$M. The activity was inhibited by pyrophosphate anions and to a lesser extent by sulfate anions. These results differentiate PP-PNK from other mammalian polynucleotide kinases.