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.The human genome encodes two highly related serine-threonine kinases, nuclear Dbf2-related kinase 1 (NDR1) and NDR2, which belong to a subfamily of kinases with roles in cell division and cell morphology (22). The two kinases represent a subclass of the AGC family of protein kinases and are partially related to protein kinase B (PKB) and protein kinase C (PKC). Human NDRs, as well as their yeast counterpart Dbf2, can be dramatically activated through inhibition of protein phosphatase 2A (PP2A) by okadaic acid (OA), demonstrating that NDR kinases require phosphorylation for activation (13,16). Indeed, NDR1 has been shown to become autophosphorylated on Ser281 in a Ca 2ϩ -dependent manner (23), while Thr444 is phosphorylated by a hydrophobic motif kinase (M. R. Stegert et al., submitted for publication). Phosphorylation on both residues is essential for full activation of NDR1 in vitro and in vivo (16,23). Recently, the phosphorylation of NDR2 on Ser282 and Thr442 was also shown to be required for full activation of enzymatic activity (20).The founding member of the Mps1 one binder (MOB) family is the yeast protein MOB1p (12), a molecule functioning as a coactivator of yeast Dbf2p and Dbf20p (10,11,13). As previously published, human MOB1A (hMOB1A) binds NDR1 and NDR2 (NDRs) at their N termini, leading to kinase activation in vitro (3). In addition, hMOB1B and hMOB2 also stimulated NDR activity in vitro (4). However, while the MOB regulation of the NDR kinases seems to be conserved from yeast to humans, the function(s) of mammalian NDRs is unknown. Although the basic mechanism of activation of human NDR protein kinase in vitro has been described in detail (3,16,20,23), the regulation of NDR in a cellular context still remains to be elucidated. Such information should bring insight into its cellular functions and, in particular, into its possible role ...

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 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

“…Evidence has shown that hMOB1A and hMOB1B bind with LATS1/2 and NDR1/2 [45,[62][63][64][65][66][67][68][69][70]. However, it is unclear whether other members of the human MOB family also interact with LATS and NDR kinases.…”

“…hMOB1B has been shown to bind to and activate the kinase activities of LATS1, LATS2, NDR1 and NDR2 at the plasma membrane upon phosphorylation by mammalian sterile-20 like (MST) kinases ( Figure 5) [23,45,[62][63][64][65][66]. hMOB1B interacts with NDR protein kinases at their conserved N-terminal regulatory domain, also known as the S100B-MOB association (SMA) domain.…”

“…Since hMOB1A and hMOB1B share 97% identity and hMOB1B has been widely studied [45,[61][62][63][64][65][66], hMOB1B was used in our experiment to examine its effects on tumor cell growth in vivo. Additionally, membrane-targeting of hMOB1 has been demonstrated to increase LATS1 kinase activity dramatically [66], therefore, we also examined Figure 12.…”