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

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.NDR 1 kinase belongs to a highly conserved family of kinases, a subclass of the AGC family of protein kinases (1, 2). The NDR family consists of the mammalian protein kinases NDR1 and NDR2, Drosophila melanogaster NDR, Caenorhabditis elegans SAX1, mammalian, D. melanogaster and C. elegans large tumor suppressor kinases, Neurospora crassa COT1, Ustilago maydis UKC1, Saccharomyces cerevisiae Cbk1, Dbf2, and Dbf20, Schizosaccharomyces pombe Orb6 and Sid2, and several plant kinases (1, 2). These kinases share a high sequence conservation, and some possess conserved functions, mainly involving regulation of cell morphology and the cell cycle (2-15).The kinase domain sequence of NDR is related to that of other members of the AGC group of kinases, e.g. protein kinases A, B, C, and G, PRK, p70 S6K , p90 RSK , and phosphoinositide-dependent kinase 1 (1). NDR contains all 12 subdomains of the kinase catalytic domain as described by Hanks and Hunter (16). However, the catalytic domains of all of the members of the NDR family are interrupted by an insert of 30 -60 amino acids between subdomains VII and VIII. This inserted sequence is not well conserved but is always rich in the basic amino acids, arginine and lysine. The catalytic domain insert has been shown to act as a non-consensus nuclear localization signal in the case of NDR1. NDR1 localizes predominantly to the nucleus in COS-1 cells, whereas mutant NDR1 with a deletion in the insert is localized to the cytosol (17). An additional special feature of the NDR family of kinases is a highly conserved N-terminal domain. 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 Ca 2ϩ -binding protein, S100B (18). Finally, the Cterminal extension of NDR kinase contains a broadly conserved hydrophobic motif ...

DrosophilaMob Family Proteins Interact with the Related Tricornered (Trc) and Warts (Wts) Kinases

“…Evidence for a direct physical interaction has been reported for Ndr and Mob family members from yeast, flies and mammals (Colman-Lerner et al, 2001;Mah et al, 2001;Weiss et al, 2002;Hou et al, 2003;Bichsel et al, 2004;Lai et al, 2005). Previous yeast two-hybrid experiments showed evidence for a physical interaction between Trc and Mats (Giot et al, 2003) and Mats and Wts (Lai et al, 2005).…”

Threonine 74 of MOB1 is a putative key phosphorylation site by MST2 to form the scaffold to activate nuclear Dbf2-related kinase 1

“…Their results with ours indicate that the phosphorylation of Mats/MOB1 by MSTs is conserved in human and fly. The molecular activation mechanism of NDR kinases is well studied in vitro and in vivo (Bichsel et al, 2004;Hergovich et al, 2005;Stegert et al, 2005Stegert et al, , 2006a. However, because of the presence of many isoforms, there is no report to directly discuss the involvement of MST2 and MOB1 in the activation of NDR1.…”

“…, MST2 and NDR1 form a tripartite complex, and the okadaic acid treatment enhances this formation MOB1 binds NDR1 and 2 and activates them (Bichsel et al, 2004). MOB1 and MST3 co-operatively activate NDR2 .…”

“…MST2 phosphorylates LATS1 and 2 and activates them. MOB1 also binds NDR1 and 2 and induces the autophosphorylation (Bichsel et al, 2004;Hergovich et al, 2005). MST3 phosphorylates and activates NDR2 but fails to activate the MOB1-binding deficient NDR2 mutant (Stegert et al, 2005).…”

Molecular characterization of human homologs of yeast MOB1

“…By over-expressing or suppressing human NDR1 by siRNA, the kinase has been shown to regulate centrosome duplication during the cell cycle [43]. Though several proteins including hMOB1 and S100B (a calcium binding protein) have been found to bind to and activate NDR, the significance of their interactions still remains unclear [44,45]. Moreover, there is no direct evidence that NDR is involved in human cancers.…”