Although the involvement of protein kinase C (PKC) in the activation of the mitogen-activated protein (MAP) kinase pathway has been implicated through experiments using 12-O-tetradecanoylphorbol-13-acetate (TPA), there has been no direct demonstration that PKC activates the MAP kinase pathway. A Raf-dependent intact cell assay system for monitoring the activation of MAPK/ERK kinase (MEK) and extracellular signal-related kinase (ERK) permitted us to evaluate the role of PKC isotypes in MAP kinase activation. Treatment of cells with TPA or epidermal growth factor resulted in the activation of MEK and ERK. The activation of the MAP kinase pathway triggered by epidermal growth factor was completely inhibited by dominant-negative Ras (RasN17), whereas the activation triggered by TPA was not, consistent with previous observations. The introduction of an activated point mutant of PKCδ, but not PKCα or PKCϵ, resulted in the activation of the MAP kinase pathway. The activation of MEK and ERK by an activated form of PKCδ requires the presence of c-Raf and is independent of RasN17. These results demonstrate that activation of PKCδ is sufficient for the activation of MEK and ERK and that the pathway operates in a manner dependent on c-Raf and independent of Ras.

A family of inner nuclear membrane proteins is implicated in gene regulation by interacting with chromatin, nuclear lamina and intranuclear proteins; however, the physiological functions of these proteins are largely unknown. Using a Xenopus expression screening approach with an anterior neuroectoderm cDNA library, we have identified an inner nuclear membrane protein, XMAN1, as a novel neuralizing factor that is encoded by the Xenopus ortholog of human MAN1. XMAN1 mRNA is expressed maternally, and appears to be restricted to the entire ectoderm at the early gastrula stage, then to the anterior neuroectoderm at the neurula stage. XMAN1 induces anterior neural markers without mesoderm induction in ectodermal explants, and a partial secondary axis when expressed ventrally by dorsalizing the ventral mesoderm. Importantly, XMAN1 antagonizes bone morphogenetic protein (BMP) signaling downstream of its receptor Alk3, as judged by animal cap assays, in which XMAN1 blocks expression of downstream targets of BMP signaling (Xhox3 and Msx1), and by luciferase reporter assays, in which XMAN1 suppresses BMP-dependent activation of the Xvent2 promoter. Deletion mutant analyses reveal that the neuralizing and BMP-antagonizing activities of XMAN1 reside in the C-terminal region, and that the C-terminal region binds to Smad1, Smad5 and Smad8, which are intracellular mediators of the BMP pathway. Interference with endogenous XMAN1 functions with antisense morpholino oligos leads to the reduction of anterior neuroectoderm. These results provide the first evidence that the nuclear envelope protein XMAN1 acts as a Smad-interacting protein to antagonize BMP signaling during Xenopus embryogenesis.

Signal-dependent activation of the transcription factor NF-κB is dominantly regulated by degradation of IκB-α protein. However, the signaling pathways that lead to the degradation are not clear. Here we report that mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK) kinase, an activator of stress-activated protein kinases/jun kinase-1 (SAPKs/JNK1), is involved in such signaling pathways. The transient overexpression of MEK kinase in NIH3T3 fibroblasts activates κB-CAT reporter expression in a synergistic manner with TNFα stimulation. In contrast, overexpression of kinase-negative MEK kinase suppresses TNFα-induced reporter expression. The overexpression of MEK kinase suppresses the inhibitory activity of co-transfected IκB-α on the κB-CAT or human immunodeficiency virus-long terminal repeat-luciferase reporter expression and causes the simultaneous disappearance of the overexpressed IκB-α. The disappearance of exogenous IκB-α by the overexpression of MEK kinase is prevented by calpain inhibitor-I, an inhibitor of IκB-α degradation. These results suggest that MEK kinase is a signal mediator involved in TNFα-induced NF-κB activation and that the activation of NF-κB by MEK kinase is regulated through the degradation of IκB-α.