Greatwall is a novel serine/threonine kinase essential for promoting a correct timing of mitosis (6
). The activation of this kinase is required at mitotic entry to promote the inhibition of PP2A, the phosphatase responsible for cyclin B-Cdc2 substrate dephosphorylation, whereas the inactivation of Gwl is crucial to allow a subsequent reactivation of this phosphatase and the dephosphorylation of mitotic substrates promoting mitotic exit (27
). Consequently, the correct timing of Gwl activation and inactivation is fundamental for promoting correct cell division.
Little is known about the mechanisms promoting Gwl activation: only that Gwl is phosphorylated during mitosis and that this phosphorylation correlates with the activation of this kinase.
In this study we analyzed the mechanisms responsible for Gwl activation. We show that although this is a unique kinase containing a very long insertion between catalytic domains VII and VIII, it behaves as a classical serine/threonine kinase, with functional conserved basic structural residues.
We also demonstrate that most of the AGC conserved residues of the CLA, CLT, and AST domains are functional in Gwl. However, for most AGC kinases, two different phosphorylations are required for their activation, phosphorylation of the activation loop and phosphorylation of the hydrophobic motif (12
), and neither of these two phosphorylations appears to be required for Gwl activation.
In this regard, Gwl contains a very long insert (557 amino acids) between catalytic domains VII and VIII, the classical location of the T loop in other AGC kinases. This insert could correspond to a T loop; however, two different findings call this fact into question. First, its length does not correspond to the conventional T loop of 20 to 60 amino acids. Second, the phosphorylation of a particular residue of this insert does not appear to be essential for Gwl activity. Accordingly, the point mutation or deletion of all the phospho-residues in this insert did not perturb kinase functionality. Thus, it is unlikely that Gwl could be regulated by a phosphorylation of the T loop.
Apart from the phosphorylation of the T-loop and the hydrophobic motif, most AGC kinases are phosphorylated on the tail/linker site in the AST region. This phosphorylation has been proposed in PKBβ to promote the binding of the AST site with the tail/linker binding sites in the N-terminal part of the sequence, increasing the local concentration of the hydrophobic motif in the vicinity of the hydrophobic pocket and stimulating their binding, a binding that is essential for maintaining kinase activation (16
). Gwl has a conserved tail/linker site (S875) and conserved tail/linker binding sites at the N terminus (K48 and K65). Moreover, our molecular modeling suggests an interaction of the phosphate of S875 with K48 and K65. Our phosphomapping results indicate that S875 is phosphorylated, and our mutagenic results clearly show that these interactions take place and are essential for kinase activity. It is likely that these interactions could stabilize the αC-helix to promote correct kinase activation. In addition, the presented data demonstrate that the interaction of the tail/linker site with the tail/linker binding site protects phosphorylation of the tail/linker amino acid, since when this interaction is disrupted by mutagenesis, S875 phosphorylation decreases. Moreover, dephosphorylation of S875 correlates with the decrease of kinase activity, a fact that confirms the hypothesis that phosphorylation of S875 is essential for the stabilization of the active kinase.
Another particular feature of Gwl is that, unlike most AGC kinases, it does not display a conventional hydrophobic motif, although, as it has been demonstrated for PDK1, it has a functional hydrophobic pocket. PDK1 displays the intrinsic ability to phosphorylate its own T-loop residue and thus possesses a basal kinase activity (7
). It has also been shown that the binding of the hydrophobic pocket of PDK1 to the phosphorylated hydrophobic motif of their substrates promotes conformational changes in the PDK1 catalytic core. These changes promote a severalfold increase of activity of this kinase and the phosphorylation of their substrates in their T loop (3
). Our results show that, similarly to PDK1, a synthetic peptide encompassing the carboxy-terminal hydrophobic motif of Rsk2 stimulates Gwl activity, suggesting that the binding of the hydrophobic pocket of Gwl to the phosphorylated hydrophobic motif of other AGC kinases participates in the activation of this kinase, likely by promoting conformational changes in its catalytic core. However, this activation takes place only if the tail/linker residue of Gwl (S875) has previously been phosphorylated.
Thus, although Gwl belongs to the AGC kinases, it appears to be regulated differently from the rest of the members of this family. Our results give key insights into the mechanisms that regulate Gwl activation. We propose that two different steps are required to promote this activation. The first step engages the phosphorylation of the tail/linker residue (residue S875). This phosphorylation promotes the binding of the phosphate of this residue with the tail/linker-binding site at the N terminus of Gwl, stabilizing the αC-helix in a partially active form. The second step involves the association of the hydrophobic pocket of Gwl to the phosphorylated hydrophobic motif of another AGC kinase, resulting in the complete activation of Gwl. In this model, the kinase responsible for the phosphorylation of the tail/linker site would be critical for promoting the correct timing of mitosis. We do not know which kinase plays this role in vivo; however, we know that the tail/linker site can be phosphorylated in vitro by both Plx1 and by cyclin B-Cdc2. It is possible that cyclin B-Cdc2 phosphorylates Gwl at mitotic entry. This phosphorylation could then participate in a positive feedback in which the accumulation of the cyclin B-Cdc2 complex could results in a partial activation of Gwl that through the inhibition of PP2A could participate in Cdc25 activation and Myt1/Wee1 inhibition, triggering the cyclin B-Cdc2 amplification loop. However, we cannot exclude the possibility that Plx1, already active in prior G2 cyclin B-Cdc2, could also be the primary kinase that activates Gwl by S875 phosphorylation. Once Gwl is activated, cyclin B-Cdc2 could maintain the tail/linker phosphorylation and thereby ensure and maintain the activation of Gwl until cyclin B degradation at mitotic exit.