Human NDR Kinases Are Rapidly Activated by MOB Proteins through Recruitment to the Plasma Membrane and Phosphorylation

Abstract

Human nuclear Dbf2-related kinases (NDRs) are up-regulated in certain cancer types, yet their precise function(s) and regulatory mechanism(s) still remain to be defined. Here, we show that active (phosphorylated on Thr444) and inactive human NDRs are both mainly cytoplasmic. Moreover, NDR kinases colocalize at the plasma membrane with human MOBs (hMOBs), which are recently described coactivators of human NDR in vitro. Strikingly, membrane targeting of NDR results in a constitutively active kinase due to phosphorylation on Ser281 and Thr444 that is further activated upon coexpression of hMOBs. Membrane-targeted hMOBs also robustly promoted activation of NDR. We further demonstrate that the in vivo activation of human NDR by membrane-bound hMOBs is dependent on their interaction and occurs solely at the membrane. By using a chimeric molecule of hMOB, which allows inducible membrane translocation, we found that NDR phosphorylation and activation at the membrane occur a few minutes after association of hMOB with membranous structures. We provide insight into a potential in vivo mechanism of NDR activation through rapid recruitment to the plasma membrane mediated by hMOBs.

SUPPLEMENTAL MATERIAL

Supplemental material for this article may be found at http://mcb.asm.org/.

ACKNOWLEDGMENTS

We thank all members of our laboratory for helpful discussions. We also thank M. Pietrzak (Friedrich Miescher Institute) for DNA sequencing, F. Fischer (Friedrich Miescher Institute) for synthesis of peptides, and J. Rohrer (University of Zurich) for anti-CLIMP63 antibody. Special thanks go to P. A. Silver (Dana-Farber Cancer Institute) for providing Flag-tagged hMOB1B and hMOB2 cDNAs. We are grateful to J. Lisztwan for careful reading of the manuscript.

This work was supported by Swiss Cancer League grant KLS-01342-02-2003 (to B.A.H.), and A. Hergovich was supported by a fellowship of the Roche Research Foundation.

The Friedrich Miescher Institute is part of the Novartis Research Foundation.