Identification of a Wee1–Like Kinase Gene Essential for Procyclic Trypanosoma brucei Survival

Abstract

Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.

Coordination of the Cell Cycle in Trypanosomes

“…The G 2 to M DNA damage checkpoint in yeast requires, among others, Chk1, Wee1, and Cdc25, which do not have unambiguous homologs in trypanosomes. However, a Wee1-like kinase is required for trypanosome cell cycle progression (18). Therefore, we cannot exclude a checkpoint at the G 2 to M transition.…”

Profiling the bloodstream form and procyclic form Trypanosoma brucei cell cycle using single-cell transcriptomics

“…In late G1, several genes associated with ‘protein phosphorylation’ are evident, including known cell cycle-associated genes such as CRK2 ( Mottram and Smith, 1995 ; Tu and Wang, 2005 ; Tu and Wang, 2004 ), aurora kinase 3 (AUK3; Jones et al, 2014 ; Tu et al, 2006 ), and Wee1-like kinase ( Boynak et al, 2013 ). Fourteen late G1 genes related to the term ‘DNA replication’: 5 genes encoding components of the MCM DNA replication licensing complex (MCM2, 4–7), an MCM10 homolog, 5 DNA polymerases, 2 DNA primase subunits and the DNA synthesis factor RNR1 (ribonucleoside-diphosphate reductase large chain).…”

Profiling the bloodstream form and procyclic formTrypanosoma bruceicell cycle using single cell transcriptomics

“…All GO terms, including "rRNA processing", relate to one gene, Tb427_110120100, which shares sequence homology with UTP21, a component of the small-subunit processome 32 . In late G1, several genes associated with "protein phosphorylation" are evident, including known cell cycle associated genes such as CRK2 10,33,34 , aurora kinase 3 (AUK3 35,36 ) and Wee1-like kinase 37 . 14 late G1 genes related to the term "DNA replication": 5 genes encoding components of the MCM DNA replication licensing complex (MCM2, 4-7), an MCM10 homolog, 5 DNA polymerases, two DNA primase subunits and the DNA synthesis factor RNR1 (ribonucleoside-diphosphate reductase large chain).…”