Phosphoproteome analysis of the near-haploid cell line HAP1 reveals phosphorylation events originating from PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK)
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Medientyp:
E-Book
Titel:
Phosphoproteome analysis of the near-haploid cell line HAP1 reveals phosphorylation events originating from PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK)
Erschienen:
[Erscheinungsort nicht ermittelbar]: [Verlag nicht ermittelbar], 2021
Sprache:
Englisch
Entstehung:
Hochschulschrift:
Dissertation, 2021
Anmerkungen:
Beschreibung:
PDZ-binding kinase/T-LAK cell-originated protein kinase (PBK/TOPK) is a serine/threonine kinase, phylogenetically related to mitogen-activated protein kinase kinase (MAPKK). PBK has a low abundance on the protein level in differentiated cells, while its abundance and activity increase in fetal cells and various malignant cells. During the G2/M transition, PBK has been reported to phosphorylate the inter-zinc finger linker sequence (TGEKP) of hundreds of C2H2 zinc finger proteins implicated in deliberating these transcription factors from the condensing chromatin. This study aimed to interrogate PBK-regulated proteome and investigate PBK-dependent phosphorylation events by analyzing the proteome and the phosphoproteome of both wild and PBK knockout HAP1 cells. The near-haploid human cell line HAP1 was adopted as a model to identify PBK-dependent phosphorylation events due to the availability of wild type and PBK knockout cell lines. The wild type and PBK knockout HAP1 cells were either arrested in the mitotic phase or allowed to proliferate exponentially, representing four conditions. PBK activity was then evaluated by examining PBK phosphorylation specific substrate (TGEKP) using monoclonal antibodies. Flow cytometry was used to characterize the cell cycle phases. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) was used to acquire the proteome and phosphoproteome of the analyzed conditions. The proteome analysis showed 20 proteins being downregulated and one protein (GPC4) upregulated during the mitotic phase. A total of 39 proteins had significantly different abundances among the four analyzed conditions. Besides PBK kinase, only three proteins displayed different expression levels between mitotic wild type and PBK knockout cells. Western blot validation of three differentially regulated proteins (ANXAI, G6PD, and EMD) revealed that protein abundances vary among different HAP1 clones, which have the same genotype. Due to the variability of protein abundances among clones with the same genotype, ...