Nemo-Like Kinase,an Essential Effector of Anterior Formation,Functions Downstream of p38 Mitogen-Activated Protein Kinase

Nemo-like kinase (NLK) is known to function as a mitogen-activated protein kinase (MAPK)-like kinase. However, the upstream molecules and molecular mechanisms that regulate NLK activity remain unclear. In the present study, we identified p38 MAPK as an upstream kinase and activator of NLK. p38 regulates the function of NLK via phosphorylation, and this modification can be abrogated by depletion of endogenous p38. In Xenopus laevis embryos, depletion of either p38β or NLK by antisense morpholino oligonucleotides results in a severe defect in anterior development and impaired expression of endogenous anterior markers. It is notable that morphants of Xenopus p38α, another isoform of the p38 MAPK family, exhibited no obvious defects in anterior development. Defects in head formation or in the expression of anterior marker genes caused by suppression of endogenous p38β expression could be rescued by expression of wild-type NLK but not by expression of mutant NLK lacking the p38β phosphorylation site. In contrast, defects in head formation or in the expression of anterior marker genes caused by suppression of endogenous NLK expression could not be rescued by expression of p38. These results provide the first evidence that p38 specifically regulates NLK function, which is required for anterior formation in Xenopus development.Nemo-like kinase (NLK) is an evolutionarily conserved serine-threonine protein kinase that was originally isolated as a murine orthologue of Drosophila melanogaster Nemo, which is involved in diverse signaling processes (3). Studies of Nemo-null mutants in Drosophila revealed that Nemo plays a role in head development and in the pathway governing epithelial planar cell polarity during eye development by controlling programmed cell death (19). In our previous studies, we demonstrated that NLK is involved in the suppression of the Wnt/β-catenin signaling pathways. NLK inactivates a transcriptional unit composed of β-catenin/T-cell factor (TCF)/lymphoid enhancer-binding factor (LEF) by phosphorylation of TCF/LEF, which inhibits the binding of this complex to its target gene sequences (10, 28). NLK functions downstream of transforming growth factor β-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK or MAP3K) family (10, 22), Wnt1 (9), and Wnt5a (8). Loss of NLK/Nemo function results in an embryonic lethal phenotype in Drosophila (19), Caenorhabditis elegans (24), and mice (15), strongly implicating NLK/Nemo as a very important regulator of cell growth, patterning, and death. We previously demonstrated that in Xenopus laevis embryos, expression of NLK is restricted to the central nervous system, eye field, and anterior neural crest cell populations. Xenopus NLK is involved in anterior formation and the expression of anterior neural marker genes (6). Our recent data indicate that, in addition to TCF/LEF, NLK associates with and modulates the activities of other transcription factors, including xSox11, STAT3 (22), HMG2L1 (27), and MEF2A (26). This suggests that NLK contributes to various signaling pathways via its ability to interact with a diverse collection of transcription factors.The activation of p38 in response to a wide range of extracellular stimuli is reflected in the diverse range of MAP3Ks (TAK1, ASK1, DLK, and MEKK4, etc.) that participate in p38 activation, illustrating the complexity of this signaling pathway (16, 17). The MAP3Ks phosphorylate and activate the MAPK kinases (MAP2Ks) MKK6 and MKK3, which in turn phosphorylate the p38 MAPKs. In vertebrates, there are four isoforms of p38: p38α, p38β, p38γ, and p38δ. These isoforms are characterized by a Thr-Gly-Tyr (TGY) dual-phosphorylation motif (11). Once activated, p38s phosphorylate their substrates on serine/threonine residues. The list of reported downstream substrates of p38 continues to expand and includes other protein kinases and many transcription factors, suggesting its possible role in regulating gene expression at the transcriptional level. Analysis of several of the downstream targets of p38 that are lineage specific or that play an essential role in development have indicated a central role of the p38 pathway in various developmental and differentiation processes (21).In the present study, we report the novel finding that the p38β isoform is a functional partner of NLK. NLK was found to associate with, and to be specifically phosphorylated by, p38β. Depletion of either Xenopus p38β (xp38β) or xNLK resulted in defects in anterior neural development in Xenopus embryos, including the loss of eye and head structures. The phenotypes induced by depletion of endogenous xp38β were rescued by overexpression of wild-type xNLK but not by a nonphosphorylatable mutant of xNLK. These results reveal a new role of p38β in the phosphorylation and regulation of NLK function during anterior formation.