Functional characterisation of the mammalian NDR1 and NDR2 protein kinases and their regulation by the mammalian Ste20-like kinase MST3

Protein modification is a common regulatory mechanism in order to transduce a signal from
one molecule to another. One of the best-studied protein modifications is phosphorylation.
The enzymes that are capable of transferring phosphate groups onto other proteins are called
protein kinases. Depending on the acceptor group, kinases can be distinguished into tyrosine,
serine/threonine and dual-specificity kinases. This work describes the characterisation of
human and mouse NDR1 and NDR2 kinases, members of the AGC group of serine/threonine
kinases. The NDR protein kinase family is highly conserved between yeast and human, and
several members have been shown to be involved in the regulation of cell morphology and the
control of cell cycle progression. For example, the yeast NDR kinases Sid2p
(Schizosaccharomyces pombe) and Dbf2p (Saccharomyces cerevisiae) are central
components of the septation-initiation network and the mitosis exit network, respectively. The
closest yeast relatives Cbk1p and Orb6p, members of the regulation of Ace2p transcription
and morphogenesis network and Orb6 signalling pathways, are implicated in the coordination
of cell cycle progression and cell morphology. This study, as well as studies using worms and
flies, provide evidence that not only NDR is conserved, but also the NDR signalling pathway
and regulation. Similar to yeast, NDR kinase activation is regulated by phosphorylation at the
activation segment phosphorylation site and the hydrophobic motif phosphorylation site. This
phosphorylation is regulated by a conserved signaling module consisting of MOB proteins
and a STE20–like kinase. Here we show that the STE20-like kinase MST3 activates NDR by
phosphorylation specifically at the hydrophobic motif in vitro and in vivo. Furthermore,
MOB1A binding is important for the release of autoinhibition and full kinase activation. The
data also indicate that NDR is part of a feedback mechanism, which induces cleavage and
nuclear translocation of MST3. The data presented here also show that NDR1 and NDR2 are
differentially expressed, but regulated in a similar manner. Mouse Ndr1 mRNA is mainly
expressed in spleen, thymus and lung, whereas Ndr2 mRNA is more ubiquitously expressed,
with the highest levels in the gastrointestinal tract. Both, NDR1 and NDR2, are activated by
S100B protein and okadaic acid stimulated phosphorylation; NDR1 and NDR2 are also
indistinguishable in the biochemical assays used: membrane targetting, phosphorylation by
MST3, and activation by MOB. Further, this work describes the generation and initial
characterisation of a mouse model for NDR1 deficiency. Protein analysis using NDR1
knockout mouse embryonic fibroblasts suggest a compensation of the loss of NDR1 by
upregulation of NDR2 expression.