Shizuka Ishitani

Nemo-like kinase suppresses Notch signalling by interfering with formation of the Notch active transcriptional complex

The Notch signalling pathway has a crucial function in determining cell fates in multiple tissues within metazoan organisms. On binding to ligands, the Notch receptor is cleaved proteolytically and releases its intracellular domain (NotchICD). The NotchICD enters the nucleus and acts cooperatively with other factors to stimulate the transcription of target genes. High levels of Notch-mediated transcriptional activation require the formation of a ternary complex consisting of NotchICD, CSL (CBF-1, suppressor of hairless, LAG-1) and a Mastermind family member. However, it is still not clear how the formation of the ternary complex is regulated. Here we show that Nemo-like kinase (NLK) negatively regulates Notch-dependent transcriptional activation by decreasing the formation of this ternary complex. Using a biochemical screen, we identified Notch as a new substrate of NLK. NLK-phosphorylated Notch1ICD is impaired in its ability to form a transcriptionally active ternary complex. Furthermore, knockdown of NLK leads to hyperactivation of Notch signalling and consequently decreases neurogenesis in zebrafish. Our results both define a new function for NLK and reveal a previously unidentified mode of regulation in the Notch signalling pathway.

NLK positively regulates Wnt/β-catenin signalling by phosphorylating LEF1 in neural progenitor cells

Nemo‐like kinase (NLK/Nlk) is an evolutionarily conserved protein kinase involved in Wnt/β‐catenin signalling. However, the roles of NLK in Wnt/β‐catenin signalling in vertebrates remain unclear. Here, we show that inhibition of Nlk2 function in zebrafish results in decreased Lymphoid enhancer factor‐1 (Lef1)‐mediated gene expression and cell proliferation in the presumptive midbrain, resulting in a reduction of midbrain tectum size. These defects are related to phosphorylation of Lef1 by Nlk2. Thus, Nlk2 is essential for the phosphorylation and activation of Lef1 transcriptional activity in neural progenitor cells (NPCs). In NPC‐like mammalian cells, NLK is also required for the phosphorylation and activation of LEF1 transcriptional activity. Phosphorylation of LEF1 induces its dissociation from histone deacetylase, thereby allowing transcription activation. Furthermore, we demonstrate that NLK functions downstream of Dishevelled (Dvl) in the Wnt/β‐catenin signalling pathway. Our findings reveal a novel role of NLK in the activation of the Wnt/β‐catenin signalling pathway.

Nemo-like kinase, a multifaceted cell signaling regulator.

Nemo-like kinase (NLK) is an evolutionarily conserved MAP kinase-related kinase. Although NLK was originally identified as a Drosophila gene affecting cell movement during eye development, recent studies show that NLK also contributes to cell proliferation, differentiation, and morphological changes during early embryogenesis and nervous system development in vertebrates. In addition, NLK has been reported to be involved in the development of several human cancers. NLK is able to play a role in multiple processes due to its capacity to regulate a diverse array of signaling pathways, including the Wnt/β-catenin, Activin, IL-6, and Notch signaling pathways. Although the molecular mechanisms that regulate NLK activity remain unclear, our recent research has presented a new model for NLK activation. Here, we summarize the current understanding of the function and regulation of NLK and discuss the aspects of NLK regulation that remain to be resolved.

Nemo-like kinase is involved in NGF-induced neurite outgrowth via phosphorylating MAP1B and paxillin

Nerve growth factor (NGF) promotes neurite outgrowth through regulating cytoskeletal organization and cell adhesion. These activities are modulated by protein phosphorylation. Nemo‐like kinase (NLK) is an evolutionarily conserved MAP kinase‐like kinase that phosphorylates several transcription factors. Although NLK is known to be expressed at relatively high levels in the nervous system, its function is not well understood. We found that NGF promotes the translocation of NLK to PC12 cells’ leading edges, and triggers NLK kinase activity in them. Activated NLK directly phosphorylates microtubule‐associated protein‐1B (MAP1B) and the focal adhesion adaptor protein, paxillin. Knockdown of NLK attenuates the phosphorylation of both paxillin and MAP1B and inhibits both the NGF‐induced re‐distribution of F‐actin and neurite outgrowth. We also discovered that NLK is a LiCl‐sensitive kinase. LiCl is known to block NGF‐induced neurite outgrowth and the phosphorylation of MAP1B and paxillin in PC12 cells. Therefore, the effects of LiCl are mediated in part by blocking NLK activity. These results suggest that NLK controls the dynamics of the cytoskeleton downstream of NGF signaling.

Homodimerization of Nemo-like kinase is essential for activation and nuclear localization

NLK is an evolutionarily conserved protein kinase that phosphorylates several transcription factors. However, the molecular mechanisms that regulate NLK activity have been poorly understood. This study shows that homodimerization of NLK is required for its activation and nuclear localization.

Hipk2 and PP1c Cooperate to Maintain Dvl Protein Levels Required for Wnt Signal Transduction

The phosphoprotein Dishevelled (Dvl) is a common essential component of Wnt/β-catenin and Wnt/planar cell polarity (PCP) signaling pathways. However, the regulation and significance of Dvl phosphorylation are not fully understood. Here, we show that homeodomain-interacting protein kinase 2 (Hipk2) facilitates protein phosphatase 1 catalytic subunit (PP1c)-mediated dephosphorylation of Dvl via its C-terminal domain and that this dephosphorylation blocks ubiquitination and consequent degradation mediated by the E3 ubiquitin ligase Itch, which targets the phosphorylated form of Dvl proteins. Inhibition of Hipk2 or PP1c function reduces Dvl protein levels and suppresses Wnt/β-catenin and Wnt/PCP pathway-dependent events in mammalian cells and zebrafish embryos, suggesting that Hipk2 and PP1c are essential for maintaining Dvl protein levels that are sufficient to activate Wnt signaling. We also show that Wnt-3a, a Wnt/β-catenin ligand, induces dissociation of the Dvl-Hipk2-PP1c complex and Dvl degradation under high-cell-density conditions. This regulation may be a negative feedback mechanism that fine-tunes Wnt/β-catenin signaling.

Cell competition corrects noisy Wnt morphogen gradients to achieve robust patterning

Morphogen signaling forms an activity gradient and instructs cell identities in a signaling strength-dependent manner to pattern developing tissues. However, developing tissues also undergo dynamic morphogenesis, which may produce cells with unfit morphogen signaling and consequent noisy morphogen gradient. Here we show that a cell competition-related system corrects such noisy morphogen gradients. Zebrafish imaging analyses of the Wnt/β-catenin signaling-gradient, which acts as a morphogen to establish embryonic anterior-posterior patterning, revealed that unfit cells with abnormal Wnt/β-catenin activity spontaneously appear and produce noise in the Wnt/β-catenin-gradient. Communication between the unfit and neighboring fit cells via cadherin proteins stimulates the apoptosis of the unfit cells by activating Smad signaling and reactive oxygen species production. This unfit cell elimination is required for proper Wnt/β-catenin-gradient formation and consequent anterior-posterior patterning. Because this gradient controls patterning not only in the embryo but also in adult tissues, this system may support tissue robustness and disease prevention.

Nemo-like kinase promotes neurogenesis by interfering with formation of Notch transcription complex

O2-4-2-3 Nemo-like kinase promotes neurogenesis by interfering with formation of Notch transcription complex Tohru Ishitani 1 , Tomoko Hirao 2, Maho Suzuki 3, Miho Isoda 2, Shizuka Ishitani 1, Motoo Kitagawa 4, Kunihiro Matsumoto 3, Motoyuki Itoh 2 1 Div. of Cell Reg. Sys., M.I.B., Kyushu University, Fukuoka, Japan 2 Inst. for Advanced Research, Nagoya University, Nagoya, Japan 3 Group of Signal Transduction, Laboratory of Cell Regulation, Div. of Biol. Sci., Grad. Sch. of Sci., Nagoya University, Naguya, Japan 4 Dep. of Mol. and Tumor Path., Chiba University Grad. Sch. of Med., Chiba, Japan