Funktionelle Untersuchungen der Protein-Histidin-Phosphatase und Identifizierung der ß-Untereinheit des heterotrimeren G-Proteins als deren Substrat
Im Rahmen der hier vorliegenden Arbeit sollte die erste im Säugetier entdeckte Protein-Histidin-Phosphatase (PHP) weiter charakterisiert und ihre physiologische Funktion in der Zelle näher untersucht werden. Aus diesem Grund wurde versucht, ein geeignetes Modellsystem zu entwickeln, mit dessen Hilfe...
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Table of Contents: Within this work the physiological function of the first protein histidine phosphatase (PHP) discovered in mammals should be investigated. For that reason an appropriate model system for changing the cellular expression rate of PHP in the cell was developed to carry out functional studies. Furthermore, new substrates of PHP were searched for that might give information about the phosphatases functional properties. First, the expression of PHP in embryonic rat neurons was downregulated with the help of antisense technology. It could be demonstrated that the antisense oligonucleotides saved the neurons from dying by staurosporine-induced apoptosis. Although this protection could be achieved with different antisense oligonucleotides directed against various sections of the PHP-sequence, it could not be achieved with mismatch-oligonucleotides. Therefore, the protective effect was specific and one could assume that PHP is involved in the regulation of apoptosis in neuronal cells. Unfortunately, it was not possible to show the protective effect of the antisense oligonuceotides and the downregulation of PHP simultaneously in the same cells. Hence no direct evidence existed that the 20-30% decrease in apoptosis was attributed to the reduced amount of PHP. Nevertheless, it was striking that as investigations with fluorescence-marked oligonuceotides demonstrated only 20-30% of the neurons contained antisense oligonucleotides after treatment. This percentage correlated with the decrease of staurosporine-induced damage and supported the assumption that PHP is involved in the regulation of apoptosis. To investigate this further, a recombinant adenovirus containing the PHP-gene was constructed. Cells infected by this virus (Ad5-PHP) were able to overexpress PHP. This was also achieved in SY5Y cells, a cell line of neuronal origin. After 24 h of infection these cells showed a change in morphology, they became small and round and seemed not to tolerate the PHP-overexpression, which resulted by the infection. Investigations of apoptosis demonstrated that cells infected with Ad5-PHP were significantly more damaged than control cells infected by an adenovirus not containing the PHP-gene. Therefore, the cell death was not traced back to the virus infection itself but to the overexpression of PHP. For the first time it had been possible to reveal a function of PHP: overexpression of this enzyme leads to apoptosis in neuronal cells. In these cells PHP is involved in the regulation of programmed cell death. Further, it could be shown within this work that the beta-subunit of the heterotrimeric G-protein is a new physiological substrate of PHP. G-beta is phosphorylated at histidine-266 by nucleoside-diphosphate-kinase isoform B and is dephosphorylated by PHP. This dephosphorylation is specific for PHP. It was not observed with alkaline phosphatase. Additionally, the finding that G-beta and PHP are co-localized in tissues of various organs such as the heart, brain and lung as well as in different regions of the brain such as the cortex, cerebellum, striatum and hippocampus indicated the physiological relevance of this dephosphorylation reaction.