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)
Creators: name: Albony, Hasan
Publisher: name: Zenodo
Year: 2021
Subjects: TOPK/PBK, Mitosis, Kinase, C2H2 Zinc Finger Protein, HAP1 cells

description: 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, several individual clones have to be analyzed. Phosphoproteome analysis of mitotic and non-mitotic HAP1 cells revealed 299 upregulated and 45 downregulated phosphosites during mitosis. Motif analysis of the mitotic upregulated phosphosites showed seven consensus phosphorylation motifs (RXXpS, pSL, pS/T, pSA, pSV, and GpS). Comparing the phosphoproteomes of mitotic wild type and PBK knockout HAP1 cells showed five candidate PBK-dependent phosphosites. The identified candidates belong to DNA- and RNA binding proteins (ADNP and FUBP1) or dynamic regulators of cell organelles (MARCKS and PGRMC1). Interestingly, one of these candidates is derived from FUBP1 and has the amino acid sequence (TGADKP) that resembles a specific substrate of PBK (TGEKP). These results suggest that PBK is responsible for these phosphorylation events on ADNP, FUBP1, MARKS, and PGRMC1, either by the direct phosphorylation (direct substrates) or by phosphorylation and activation of their upstream kinases (indirect substrates). These phosphorylation events may modulate the activities of these proteins and have a vital role in cell division. Further inspection of the PBK`s impact on the identified candidate PBK-dependent phosphosites is requested to differentiate between direct and indirect PBK substrates. Besides, the investigation of these phosphosites in regulating protein functions is essential to understand the impact of PBK on modulating the involved biological processes.