This dissertation, "Molecular and Cellular Characterization of Ganglioside-stimulated Protein Kinase" by Erik Armand Jaan, Miljan, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled Molecular and Cellular Characterization of Ganglioside-stimulated Protein Kinase submitted by Erik Armand Jaan Miljan for The Degree of Doctor of Philosophy at The University of Hong Kong in May 1999 A ganglioside-stimulated serine/threonine protein kinase, termed PKJ, was purified from guinea pig brain. A nineteen amino acid tryptic peptide, containing the autophosphorylation site of PKJ, was isolated and subjected to automated amino acid sequencing. The PKJ phosphotryptic peptide sequence, SVIDPIPAPAGDSHVDGGA, is highly homologous to the mammalian p21-activated kinase (PAK) family. Moreover, the ganglioside-stimulated autophosphorylation site of PKJ aligns with a known phosphorylation site in PAKs. To confirm if the homology between PKJ and PAKs was authentic, PAK antibodies were used in cross-reactivity studies. The monospecific polyclonal antibodies, anti-PAK1(N-20), anti-PAK1(C- 19) and anti-PAK3(N-19), specific to PAK1 and PAK3 were found to cross-react with PKJ by both western blot and immunoprecipitation experiments. In addition, immunoprecipitated PAK1 and PAK3 from mouse brain were also found to undergo ganglioside-stimulated autophosphorylation in a similar manner as PKJ. These findings are in agreement with previous reports that PAK1 can be stimulated by gangliosides; however, PAK3 is shown for the first time to undergo ganglioside-stimulated GTPase- independent activation. Furthermore, both ganglioside-stimulated PAK1 and PAK3 were found to display the same physiochemical properties as previously shown for PKJ. Although it is difficult to conclude whether PKJ is a unique PAK isoform or not, this data strongly indicates that PKJ is a member of the PAK family. To gain insights into the physiological role of PKJ, anti-PAK1(N-20) was used to screen normal mouse organs. Anti-PAK1(N-20) staining was observed in all organs examined, indicating the ubiquitous nature of PAK1. Cell types were found to vary in the staining intensities. Epithelium cells displayed consistently higher levels of anti- PAK1(N-20) immunoreactivity compared to connective tissue, for example fibroblasts, whose immunoreactivity was barely detectable. Cell types with excitablemembranes were observed to have increased anti-PAK1(N-20) staining, namely muscle and nerve cell types. Cytosolic and plasma membrane staining patterns were observed and are consistent with current understanding of PAK1subcellular localization in the cell. However, anti-PAK1(N-20) punctate staining patterns were observed in many different cell types, indicating PAK1 may be associated with vesicular formations. Because gangliosides are lipids, the association between PAK1 and vesicle bodies requires further investigation. DOI: 10.5353/th_b2995761 Subjects: Protein kinasesGangliosidesCellular signal transduction