Mechanism of Activation of NDR (Nuclear Dbf2-related) Protein Kinase by the hMOB1 Protein

Samuel J Bichsel,Rastislav Tamaskovic,Mario R Stegert,Brian A Hemmings

doi:10.1074/jbc.m404542200

Samuel J Bichsel, Rastislav Tamaskovic + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m404542200

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Abstract

NDR (nuclear Dbf2-related) kinase belongs to a family of kinases that is highly conserved throughout the eukaryotic world. We showed previously that NDR is regulated by phosphorylation and by the Ca(2+)-binding protein, S100B. The budding yeast relatives of Homo sapiens NDR, Cbk1, and Dbf2, were shown to interact with Mob2 (Mps one binder 2) and Mob1, respectively. This interaction is required for the activity and biological function of these kinases. In this study, we show that hMOB1, the closest relative of yeast Mob1 and Mob2, stimulates NDR kinase activity and interacts with NDR both in vivo and in vitro. The point mutations of highly conserved residues within the N-terminal domain of NDR reduced NDR kinase activity as well as human MOB1 binding. A novel feature of NDR kinases is an insert within the catalytic domain between subdomains VII and VIII. The amino acid sequence within this insert shows a high basic amino acid content in all of the kinases of the NDR family known to interact with MOB proteins. We show that this sequence is autoinhibitory, and our data indicate that the binding of human MOB1 to the N-terminal domain of NDR induces the release of this autoinhibition.

Highlights

Other members of the AGC group of kinases, e.g. protein kinases A, B, C, and G, PRK, p70S6K, p90RSK, and phosphoinositide-dependent kinase 1 [1]
In the case of NDR1, this domain consists of 81 amino acids and encompasses a region predicted to form an amphiphilic ␣-helix that binds to the EF-hand Ca2ϩ-binding protein, S100B [18]
To test whether hMOB1 plays a role in NDR activation, we co-transfected Myc-hMOB1 or the empty vector with HA-NDR1 into COS-1 cells

Summary

The kinase domain sequence of NDR is related to that of

We have shown that Ser-281 is autophosphorylated, whereas Thr-444 is targeted by an as yet unidentified upstream kinase Both sites are crucial for NDR activity in vivo and in vitro. The results of several recent studies indicate a novel conserved signaling pathway involving NDR kinase family members. It has been shown in D. melanogaster that NDR genetically interacts with FURRY, a 300-kDa protein of unknown function [22]. The FURRY-like proteins are conserved in mammals, and it is likely that other proteins interacting genetically and/or physically with S. cerevisiae Cbk or Dbf play a role in the NDR kinase family pathway in higher eukaryotes. We show that a basic sequence within the insert in the catalytic domain of NDR has an autoinhibitory function and that hMOB1 may stimulate NDR activity by releasing the autoinhibitory effect of this sequence

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION