Dual-specificity kinase Myt1: Insights into specificity, mechanism and regulation of catalytic activities

Myt1 is a dual-specificity kinase that adds inhibitory phosphates to cyclin-dependent kinase (Cdk)/cyclin complexes, thereby contributing to regulation of the cell cycle. Together with the tyrosine kinase Wee1, Myt1 prevents premature entry into mitosis and may also regulate initiation of DNA synthesis. Myt1 is found to be phosphorylated and less active towards Cdk/cyclins in M-phase compared to interphase. In vitro, multiple kinases have been described to phosphorylate Myt1 including p90 rsk, Plx and Akt. Additionally, Myt1, like many other kinases, autophosphorylates and it is possible that this is related to phosphorylation events during M-phase. Relatively little is known about the biochemistry and regulation of Myt1 and thus we have initiated a biochemical investigation of Myt1 kinase activities and regulation thereof. We have purified a Xenopus laevis Myt1 construct containing the N-terminal and catalytic domains (XMyt1-DeltaC) and addressed its substrate specificity with various protein and artificial substrates. Importantly, the activity of this construct, appears to be similar to full-length protein purified from insect cells. The XMyt1-DeltaC construct did not have any detectable activity against artificial substrates. However, it does have activity against its natural substrates, the Cdk/cyclins, specifically phosphorylating Cdk2/cyclinA and Cdc2/cyclinB complexes and not Cdk2 monomers. To monitor the activity of Myt1 on Cdk/cyclin complexes we have developed a high-throughput fluorescent polarization (FP) assay that allows one to monitor the phosphorylation status of the Cdk/cyclin proteins. This assay is based on an antibody specific for bis-phosphorylated Cdc2/Cdk2 and a fluorescently-labeled bis-phosphorylated Cdc2/Cdk2 peptide and was used to screen 16 general kinase inhibitors against Myt1 activity. In studying Myt1 autophosphorylation, we have found evidence for an intramolecular reaction mechanism. We have identified autophosphorylation sites on Myt1 using MALDI mass spectrometry. A triple mutant of these sites and not individual mutants had a significant effect on Myt1 in vitro activity towards Cdk/cyclins. Finally, we have identified three different in vivo phenotypes of Myt1 autophosphorylation site mutants by microinjecting mRNA of Myt1 into Xenopus oocytes. Our studies demonstrate that Xenopus Myt1 autophosphorylation is important for its stability, activity, and regulation, and must be considered in any mechanism describing the regulation of Myt1.