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Protein phosphorylation plays a key role in regulating protein function and has been linked to such diseases as cancer, inflammation, and neurological disorders. The introduction of the negatively charged phosphate group can alter the protein's function through the induction of conformational changes or by inhibiting or promoting protein-protein interactions. Given that many upstream kinases of phosphorylated proteins are unknown, and that mutation of phosphorylated sites to negatively charged residues have shown variable results, another method for evaluating the effects of phosphorylation is needed. Previously, phosphotyrosine had been shown to be an effective mimic for sulfotyrosine in hirudin; therefore, we expected that sulfonation of cysteine may serve as an alternative approach to make mimics of phosphorylated serine (and possibly threonine) proteins. Sulfonation of cysteine is rapidly reversed in the presence of reducing agents; therefore, this modification can be potentially used as a redox trigger for evaluating the effects of phosphorylation.
In this dissertation MEK1 - which is enzymatically activated through phosphorylation of serine 218 and 222 - was evaluated for its ability to be chemically activated by sulfonation of cysteine 218 and / or 222 through the reaction with sodium tetrathionate and sodium sulfite. Sulfonation of cysteine 218 was shown to not only mimic phosphoserine's ability to trigger activation in MEK1, but was also demonstrated to mediate phosphorylation-dependent protein-protein interactions with the polyclonal antibody anti-MEK1 [pSpS218/222]. However, in the presence of the reducing agent DTT, the sulfonated protein's enzymatic activity was reduced to its respective basal level and it was no longer able to be recognized by anti-MEK1 [pSpS218/222]. Since many upstream kinases of phosphorylated proteins are unknown, the introduction of the thiosulfate moiety by chemical means offers endless opportunities to study the effects of phosphorylation.
Advisor: | Lee, David H. |
Commitee: | Kaplan, David, Kumar, Krishna, Whitty, Adrian |
School: | Tufts University |
Department: | Chemistry |
School Location: | United States -- Massachusetts |
Source: | DAI-B 74/10(E), Dissertation Abstracts International |
Source Type: | DISSERTATION |
Subjects: | Biochemistry |
Keywords: | Cysteine, Mimic phosphorylation, Sulfonation |
Publication Number: | 3564115 |
ISBN: | 978-1-303-12681-9 |