Autophosphorylation of Ser66 on Xenopus Myt1 is a prerequisite for meiotic inactivation of Myt1.

Myt1 is a dual-specificity kinase that contributes to the regulation of the cell cycle by adding inhibitory phosphates to the cyclin-dependent kinases (Cdk/cyclins). Myt1 is found to be phosphorylated and less active in M-phase compared to interphase. Although Myt1 can be phosphorylated by several different kinases in vitro, it is not well understood how Myt1 is regulated in vivo. Additionally, the interplay between phosphorylation by other kinases and autophosphorylation has not been investigated. Since phosphorylation is an important mode of regulation for Myt1, we have investigated the properties and physiological significance of the autophosphorylation of Myt1 from Xenopus laevis (XMyt1). Using MALDI mass spectrometry we have identified Ser66 and Ser76 as autophosphorylation sites. Autophosphorylation is important for the activity of XMyt1 in intact cells, as found by comparing the timing of the cell cycle in Xenopus oocytes expressing either exogenous wild type XMyt1 or its autophosphorylation site mutants. Specifically, S66A is significantly more potent than wild type XMyt1 at delaying entry into meiosis and concomitantly is hypophosphorylated as evident by a loss of mobility shift. However, this cannot be accounted for by a simple increase in kinase activity towards Cdk/cyclins in vitro. We therefore propose that Myt1 catalyzed autophosphorylation of residue S66 is a prerequisite and/or trigger for the further phosphorylation and inactivation of Myt1. Thus autophosphorylation of Myt1 is a novel inhibitory mechanism that adds another layer of complexity to the phosphorylation-dependent mechanism of Myt1 regulation.

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