Abstract
The Xenopus polo-like kinase Plx1 plays important roles during entry into and exit from mitosis (M phase). Previous studies revealed that Plx1 is activated by phosphorylation on serine and threonine residues, and purification of an activating enzyme from mitotic Xenopus egg extracts led to cloning and characterization of Xenopus polo-like kinase kinase (xPlkk1), which can phosphorylate and activate Plx1 in vitro. In the present study, a positive feedback loop between Plx1 and xPlkk1 was shown to result in each kinase phosphorylating and activating the other. Sequencing of radiolabeled xPlkk1 after phosphorylation by Plx1 in vitro identified three phosphorylation sites each spaced three amino acids apart, two of which have the consensus acidic-X-pSer-hydrophobic described for other polo-like kinase substrates. In addition, endogenous xPlkk1 in oocytes was phosphorylated on these sites in M phase but not in interphase. A mutant xPlkk1 in which these three amino acids were changed to alanine (xPlkk1(SA3)) was unable to be phosphorylated or activated in vitro by Plxl. Depletion of Plx1 from oocyte extracts prior to stimulation of the G(2)/M transition blocked the activation of xPlkk1, but depletion of xPlkk1 before stimulation did not block Plx1 activation. These results indicate that xPlkk1 may function downstream as a target of Plx1 rather than as an upstream activating kinase during the G(2)/M transition.
Highlights
Studies in many laboratories have shown that entry into mitosis is controlled by the activation of cyclin B/Cdc2 following dephosphorylation of Tyr-15 in Cdc2 by the dual specificity phosphatase Cdc25C
Depletion of Plx1 from oocyte extracts prior to stimulation of the G2/M transition blocked the activation of Xenopus polo-like kinase kinase 1 (xPlkk1), but depletion of xPlkk1 before stimulation did not block Plx1 activation. These results indicate that xPlkk1 may function downstream as a target of Plx1 rather than as an upstream activating kinase during the G2/M transition
Cdc25C itself is activated at the G2/M transition by phosphorylation on serine and threonine residues [1,2,3]. This activating phosphorylation can be performed by cyclin B/Cdc2 itself, evidence in Xenopus eggs and extracts and in mammalian cells suggests that initial activation of Cdc25C depends on phosphorylation by a polo-like kinase (4 –9)
Summary
Studies in many laboratories have shown that entry into mitosis is controlled by the activation of cyclin B/Cdc2 following dephosphorylation of Tyr-15 in Cdc2 by the dual specificity phosphatase Cdc25C. A positive feedback loop between Plx1 and xPlkk1 was shown to result in each kinase phosphorylating and activating the other.
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