by Yeung Shing On. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1989 / Bibliography: leaves 91-112.

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The histidine-containing protein (HPr) is a central phosphotransfer component of the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS) that transports carbohydrates across the cell membrane of bacteria. There are two HPr phosphorylation events investigated in this thesis. Firstly, BPr from Gram-positive species may undergo a regulatory phosphorylation of an absolutely conserved Ser46 residue. There are numerous metabolic consequences to this phosphorylation, including inducer exclusion and expulsion, inhibition of PTS sugar uptake and catabolite repression. While HPr from Gram-negative sources cannot undergo phosphorylation of Ser46 'in vivo' or ' in vitro' it is possible to mimic the phosphorylation through the Ser46Asp mutation. To determine the structural consequences of the mutation the crystallographic structure of the 'E. coli'. Ser46Asp HPr was determined at 1.5 Å resolution. The structure revealed that no significant structural rearrangements are induced by the mutation and the inability to accept phosphotransfer from Enzyme I is due to electrostatic disruption of the interaction of these proteins. Phosphorylation of an absolutely conserved His15 for the purpose of phosphotransfer represents the second phosphorylation event to be investigated. The absolute requirement for histidine at the 15 position was investigated through mutagenesis. The mutation of His15Asp of 'E. coli' HPr was able to accept a phosphoryl group from Enzyme I and further transfer the phosphoryl group to Enzyme IIAglc. None of the other mutations of the fifteen position were able to be phosphorylated. The His15Asp mutant had a Vmax of 0.1% and a ten-fold increase in Kin with respect to wild type HPr. As a consequence of the phosphorylation of His15Asp HPr a third protein species of higher pI than the original protein was identified. This high pI species seemed to share numerous similarities to succinimides which are known to be involved in deamidation. The inability to detect the known degradation products of succinimides suggested that the high pI species may involve isoimide formation. Isoimides have been proposed, but never experimentally demonstrated in proteins. A mechanism through which the phosphoacyl intermediate may catalyze isoimide formation is proposed. In addition the potential involvement of isoimide formation as a mechanism in physiological regulatory signaling is discussed.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Oncogenically transnformed mammalian cells irrespective of their origin synthesize and secrete osteopontin (OPN), a sialic acid rich, adhesive, phosphoglycoprotein, not only in excessive amounts but also in different molecular forms, as compared to their non-transformed counterparts. It has been postulated that OPN has important functional roles in oncogenesis, but its mechanism of action remains obscure. In the present study this question was addressed by using Rat-1 cells transformed by a temperature-sensitive mutant of Rous sarcoma virus (tsB77 cells) which secrete two discrete molecular forms of OPN, a 69-kDa OPN at the non-permissive temperature (41°C and a 62-kDa form at the permissive temperature (34°C). Investigations were aimed to determine how the two isoforms of OPN secreted by transformed and non-transformed cells originate, whether the two forms have different functional properties, and the effects of specific inhibition of OPN synthesis on the transformed state of the cells. The latter was achieved by transfecting tsB77 cells with antisense OPN cDNA at the permissive temperature. Immunoprecipitation, V8 protease mapping, tryptic peptide analysis, and thrombin digestion confirmed that 62-kDa and 69-kDa proteins are two isoforms of OPN. It was also observed that tsB77 cells at both temperatures transcribe a single 1.6 kb OPN mRNA and contain only the 69-kDa OPN intracellularly, suggesting that 69-kDa OPN is modified to its 62-kDa form prior to or immediately after secretion by cells at 34ºC. Proteolytic cleavage, differential phosphorylation, or lack of N- or O-linked carbohydrates as the possible mechanism for the generation of 62-kDa OPN were ruled out, but it was observed that 62-kDa OPN contains significantly reduced levels of sialic acid residues, as compared to its 69-kDa form. The binding assays using 32P-labeled OPN revealed that only the 69-kDa OPN, not its 62-kDa form, undergoes receptor-mediated localization on the cell surface,

Our study shows that the secretion of a major glycosylated, phosphoprotein with a molecular weight of 69kDa (pp69) is a specific marker for non-transformed NRK-49F cells. Antibody raised against pp69 recognizes, in addition to pp69, another major phosphoprotein with a molecular weight of 62kDa (pp62) secreted by RR1022 and spontaneously transformed NRK-49F cells (spt-NRK-49F). Immunoprecipitation of total cell lysates from both NRK-49F and RR1022 cells with anti-pp69 antibody detected only pp69. Treatments with: epidermal growth factor (EGF), transforming growth factor-$ beta$ (TGF-$ beta$) retinoic acid (RA), and TPA modulate the levels of pp69 present in the conditioned media. Furthermore, TPA and EGF induce the synthesis of 3 internal peptides with molecular weights of 58, 54, and 44 kDa which appear to be pre-processed forms of pp69. / Treatment of NRK-49F cells with insulin, EGF, TGF-$ beta$, PPA, levamisole and spermine clearly demonstrate alterations in the phosphorylation of pp69, concomitant with changes in extracellular phosphatase activity.

The human wildtype p53 tumor suppressor gene contains a polymorphism at amino acid residue 72 which results in either an arginine (p53 Arg-72) or proline (p53 Pro-72) at this codon. In the present study I have examined this polymorphism at the molecular level to determine whether differences exist in the biochemical functions of these two p53 variants. No differences were observed in their sequence-specific DNA binding abilities, nor in their ability to be targeted by HPV-18 E6 oncoprotein for degradation by ubiquitination in vitro. However, differences were observed in the ability of these two variants to function as transcriptional activators: p53 Pro-72 was more transcriptionally active than p53 Arg-72. I propose that the polymorphism at codon 72 may affect the structure of the N-terminal transactivation domain of the p53 protein, which would then have an effect on the ability of these variants to interact with transcription factors in order to initiate transcription of target genes and function as a tumor suppressor.

The effects of temperature on a 58-kDa phosphoprotein (PP58) have been examined in cell-free extracts of two alfalfa (Medicago sativa L.) cultivars, Apica and Trek. In the extracts prepared without the use of Triton X-100, PP58 is present in a 12,000 x g (P12), 28,000 x g (P28) and 100,000 x g (P100) pellets but is enriched in the P28 fraction. In these fractions PP58 is substantially and equally phosphorylated at both 4° and 24°C. When extracts are prepared in the presence Triton X-100, PP58 is present in the 28,000 x g supernatant (TXS fraction) is extensively dephosphorylated at 24°C but highly phosphorylated at 4°C. The phosphorylation of this protein increased sharply as temperature declined below 12°C, and was 15 times greater at 0° than at 24°C. The phosphorylation level doubled between 12° and 8°C and again between 8° and 4°C. Thus temperature effect is not mediated by Q10 effect. Interestingly, temperature-response curve of PP58 phosphorylation is similar to that of the reported cold-induced calcium influx (Plant Cell 7: 321-331). Labeling reactions carried out in the presence of [gamma-35S]thioATP indicated that low temperature inhibited the dephosphorylation reaction. These results were not mimicked at room temperature by the protein phosphatase 1 and 2A inhibitor okadaic acid. In reactions performed at 4°C, addition of calcium caused a 2-fold increase in the phosphorylation of PP58. A decrease in phosphorylation was observed when equimolar amounts of EGTA were added in the presence of MgCl 2 or MnCl2, but not in the presence of CaCl2, suggesting that this protein is phosphorylated by a calcium-dependent protein kinase. These results are consistent with the suggestion that PP58 and its putative kinase are membrane-localized whereas the putative PP58 phosphatase is a loosely-associated membrane peripheral protein lost to the supernatant during fractionation. We suggest that PP58 could be involved in low te