A Feedback Loop in the Polo-like Kinase Activation Pathway

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-pSerhydrophobic 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.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. Cdc25C itself is activated at the G 2 /M transition by phosphorylation on serine and threonine residues (1-3). Although this activating phosphorylation can be performed by cyclin B/Cdc2 itself (forming a positive feedback loop), 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). The Xenopus pololike kinase Plx1 binds Cdc25C through a noncatalytic domain, the polo box, which is thought to recognize a phosphorylated sequence in Cdc25C (10 -12). Recent evidence (13) suggests that initial phosphorylation of Cdc25C by p38␥ may facilitate binding and activation of Cdc25C by Plx1. However, prior phosphorylation is not obligatory, because Plx1 can efficiently phosphorylate bacterially expressed Cdc25C (see Fig. 2C), which presumably is not phosphorylated. Other studies on the interaction of Plx1 and Cdc25C have shown that overexpression of activated Plx1 accelerates phosphorylation of Cdc25C (14, 15), and depletion of Plx1 from oocyte extracts blocks Cdc25C activation and the G 2 /M transition (6,8). In addition to Cdc25C activation, polo-like kinase is also required for bipolar spindle formation, activation of the anaphase-promoting complex/cyclosome, and cytokinesis (14 -21). Because Plx1 controls both the activation and deactivation of cyclin B/Cdc2, it is a major player in the control of mitosis. This has focus...

Xenopus Polo-like kinase Plx1: a multifunctional mitotic kinase

“…The phosphorylation of Plx1 by xPlkk1 has been previously shown to activate Plx1 (Qian et al, 1998b), but the effect of phosphorylation of xPlkk1 by Plx1 was unknown. A subset of the sites responsible for activation was identified by a combination of direct sequencing of radiolabeled xPlkk1 and mutagenesis (Erikson et al, 2004). This study showed that Plx1 phosphorylates xPlkk1 on activating sites in vitro, and activation was additive with that due to autophosphorylation.…”

“…However, recent studies demonstrated a positive feedback loop between Plx1 and xPlkk1 (Erikson et al, 2004). In this study, it was demonstrated that Plx1 and xPlkk1 phosphorylate each other in addition to undergoing autophosphorylation (Qian et al, 1998a, b).…”

“…Mutation of these three amino acids to alanine greatly reduced the ability of Plx1 to both phosphorylate and activate xPlkk1 in vitro. A phosphospecific antibody against a synthetic peptide triply phosphorylated at these three sites recognized xPlkk1 from M phase (GVBD) oocytes, but not from G 2 phase oocytes, and also detected phosphorylation of endogenous xPlkk1 at these sites, indicating that phosphorylation at these sites occurs in vivo during the G 2 /M transition (Erikson et al, 2004).…”

Protein kinases controlling the onset of mitosis

“…The kinase responsible for Plk1 phosphorylation at the Tloop residue was initially suggested to be Xenopus Plkk1 [81], which is the homologue of human Slk. However, recent reassessment of this issue led to the conclusion that rather than being an upstream activating kinase, Plkk1 may be a target of Plk1 at the G2/M transition [82], thus leaving the issue of the mechanism of Plk1 activation still open. The close similarity of the amino acid sequence around the Plk1 T-loop site to the consensus for PDK1 led others to speculate that the latter may be a Plk1-kinase [83], though in vivo and/or in vitro evidence to support this hypothesis is not available.…”

Plk1 Regulates Activation of the Anaphase Promoting Complex by Phosphorylating and Triggering SCFβTrCP-dependent Destruction of the APC Inhibitor Emi1

“…Although some Plk1 substrates do use the chemically defined, optimal D/E-X-S/T-⌽ sequence (Nakajima et al, 2003), several mapped Plk1 phosphorylation sites do not (Yarm, 2002;Kraft et al, 2003;Erikson et al, 2004). Two of our observations-that Plk1 can phosphorylate Emi1 on serine-149, which lacks an acidic residue in the -2 position, and that mutation of glutamate-143 in front of serine-145 causes only a modest decrease in the rate of Emi1 destruction, suggest that the consensus is helpful, but not restrictive.…”