94
3.5.1.2 The AGC-Type Kinase Greatwall (Gwl)
Gwl belongs to the family of AGC-type kinases and shares many functional key resi-
dues with other family members, but it also contains an unusual nonconserved inser-
tion between the N- and the C-terminal domains (Vigneron et al. 2011 ). Gwl
activation is initiated by Cdk1-dependent phosphorylation in the conserved activa-
tion loop, which results in a conformational change that makes the active site located
between the N- and C-terminal lobes accessible (Blake-Hodek et al. 2012 ). Additional
phosphorylation events in the C-terminal domain (turn motif/tail site) are critical for
Gwl’s kinase activity. One of these C-terminal residues (S875 in human Gwl; S883 in
Xenopus Gwl) has been characterised best, and there are conflicting reports about the
kinase that mediates the phosphorylation of this residue. First, it has been deduced
from in vitro activation assays that it is phosphorylated by Cdk1 and Plk1 (Vigneron
et al. 2011 ). However, this result has been challenged by the fact that this residue gets
phosphorylated only when active, but not kinase dead, Gwl is incubated with Cdk1.
This points to an autophosphorylation mechanism, in which Gwl can phosphorylate
itself once the active site is accessible after Cdk1- dependent phosphorylation of the
activation loop (Blake-Hodek et al. 2012 ). Some AGC-type kinases, e.g. PDK1, are
only fully activated once they interact with another kinase through a hydrophobic
interaction motif. No such interaction has been described for Gwl so far, but it con-
tains a functional hydrophobic pocket. Incubation of Gwl with a phosphorylated
hydrophobic peptide of the AGC-kinase Rsk2 stimulates the kinase activity of preac-
tivated Gwl, indicating that such a transactivation mechanism with a so far unknown
partner could exist for Gwl (Vigneron et al. 2011 ). In human cell lines, Gwl is not
only regulated at the activity level but also by phosphorylation-dependent localisa-
tion. In interphase, Gwl is predominantly localised in the nucleus until it gets phos-
phorylated in the nonconserved middle region after nuclear import of Cdk1/cyclin-B
in late prophase (Alvarez- Fernandez et al. 2013 ). Upon this phosphorylation event,
Gwl is exported from the nucleus and accumulates in the cytoplasm where it medi-
ates the inactivation of PP2A-B55 before the nuclear envelope is disassembled. This
mechanism ensures that the mitotic state is not lost when nuclear Cdk1/cyclin-B and
cytoplasmic PP2A-B55 intermingle upon nuclear envelope breakdown (Alvarez-
Fernandez et al. 2013 ). In immature Xenopus laevis oocytes, which lack significant
Cdk1 activity, Gwl localisation is, in contrast, not restricted to the nucleus (Hara
et al. 2012 ). Therefore, it remains to be determined whether the mechanism of Cdk1-
mediated nuclear export of Gwl applies only to higher vertebrates or represents a
mitosis- specific mechanism.
3.5.1.3 The PP2A-B55 Inhibitors Ensa and Arpp19
In vitro assays failed to detect substantial kinase activity of Gwl towards PP2A-B55,
and this observation fuelled the search for the missing link between Gwl and
PP2A-B55 (Castilho et al. 2009 ). Two independent Xenopus laevis egg extract stud-
ies identified the two closely related proteins Arpp19 (cAMP-regulated phospho-
protein, 19 kDa) and Endosulfine α (Ensa) as the sought-after substrates of Gwl
A. Heim et al.