Dual Phosphorylation of Cdk1 Coordinates Cell Proliferation with Key Developmental Processes in Drosophila

Abstract

Eukaryotic organisms use conserved checkpoint mechanisms that regulate Cdk1 by inhibitory phosphorylation to prevent mitosis from interfering with DNA replication or repair. In metazoans, this checkpoint mechanism is also used for coordinating mitosis with dynamic developmental processes. Inhibitory phosphorylation of Cdk1 is catalyzed by Wee1 kinases that phosphorylate tyrosine 15 (Y15) and dual-specificity Myt1 kinases found only in metazoans that phosphorylate Y15 and the adjacent threonine (T14) residue. Despite partially redundant roles in Cdk1 inhibitory phosphorylation, Wee1 and Myt1 serve specialized developmental functions that are not well understood. Here, we expressed wild-type and phospho-acceptor mutant Cdk1 proteins to investigate how biochemical differences in Cdk1 inhibitory phosphorylation influence Drosophila imaginal development. Phosphorylation of Cdk1 on Y15 appeared to be crucial for developmental and DNA damage-induced G2-phase checkpoint arrest, consistent with other evidence that Myt1 is the major Y15-directed Cdk1 inhibitory kinase at this stage of development. Expression of non-inhibitable Cdk1 also caused chromosome defects in larval neuroblasts that were not observed with Cdk1(Y15F) mutant proteins that were phosphorylated on T14, implicating Myt1 in a novel mechanism promoting genome stability. Collectively, these results suggest that dual inhibitory phosphorylation of Cdk1 by Myt1 serves at least two functions during development. Phosphorylation of Y15 is essential for the premitotic checkpoint mechanism, whereas T14 phosphorylation facilitates accumulation of dually inhibited Cdk1-Cyclin B complexes that can be rapidly activated once checkpoint-arrested G2-phase cells are ready for mitosis.

Control of sleep by a network of cell cycle genes

“…We found that reduced Cdk1 activity partially rescued the short-sleeping phenotypes of tara and CycA mutants, 9 suggesting that Cdk1 is a wake-promoting molecule. Since Cdk1 is regulated through inhibitory phosphorylation of its T14 and Y15 residues, we employed a mutant Cdk1-AF (T14A, Y15F) that cannot be inhibited 24 to show that increased activity of Cdk1 suppresses sleep. Given that CycA and Cdk1 are known to physically interact, 23 a direct relationship between CycA and Cdk1 for sleep regulation is likely.…”

G2-phase arrest prevents bristle progenitor self-renewal and synchronizes cell divisions with cell fate differentiation

“…Microchaete pI cells are specified in early pupal stages but then arrest in G2 phase for almost 10 h until the mitotic cycles begin (Hartenstein and Posakony, 1989;Huang et al, 1991;Usui and Kimura, 1992). To investigate how G2 phase arrest affected SO development, we used Gal4-driven expression of a transgenic Cdk1 phospho-acceptor mutant that efficiently bypasses developmental G2 phase checkpoint arrest (Ayeni et al, 2014). Genetic crosses with a neur p72 -Gal4 strain were used to express Cdk1 Y15F -VFP (hereafter called Cdk1F) or wildtype Cdk1-VFP (as a control, hereafter called Cdk1WT) in arrested SO progenitor cells, several hours before they would normally divide (Bellaïche et al, 2001;Parks et al, 1997).…”

“…Moreover, unlike Cdk1F, overexpression of Cdc25 stg usually induced apoptosis in the Ttk + posterior pI daughter cell. In these respects, ectopic Cdc25 stg behaves more like expression of a non-inhibitable Cdk1 T14A,Y15F mutant, which causes gross chromosomal abnormalities, in addition to bypass of G2 phase checkpoint arrest (Ayeni et al, 2014). Alternatively, other factors affected by Cdc25 stg might explain this discrepancy.…”

“…The construction and phenotypic characterization of UAS-Cdk1WT and UAS-Cdk1F transgenes expressed in imaginal wing discs and larval neuroblasts was previously described (Ayeni et al, 2014). To express transgenes in the SO lineage we used either neur P72 -Gal4 (Bellaïche et al, 2001) or w; neur P72 -Gal4,P UAS mRFP1-Pon[LD][1.2]/TM3,Sb (a gift from J. Knoblich, Institute of Molecular Biotechnology, Vienna).…”

“…Expression of either a transgenic Cdk1F inhibitory phosphorylation mutant to bypass G2 phase checkpoint arrest (Ayeni et al, 2014) or Cdc25 stg to force pI cells to divide prematurely produced a secondary pI daughter cell instead of a neural precursor pIIb cell, as well as a precursor of external cells. Nonetheless, neural differentiation initiated at the normal time when the secondary pI cell divided generating an SO composed of single neuron and sheath cells, but supernumerary external cell types.…”