Hiroshi Hanafusa

Sprouty1 and Sprouty2 provide a control mechanism for the Ras/MAPK signalling pathway

Sprouty (Spry) inhibits signalling by receptor tyrosine kinases; however, the molecular mechanism underlying this function has not been defined. Here we show that after stimulation by growth factors Spry1 and Spry2 translocate to the plasma membrane and become phosphorylated on a conserved tyrosine. Next, they bind to the adaptor protein Grb2 and inhibit the recruitment of the Grb2–Sos complex either to the fibroblast growth factor receptor (FGFR) docking adaptor protein FRS2 or to Shp2. Membrane translocation of Spry is necessary for its phosphorylation, which is essential for its inhibitor activity. A tyrosine-phosphorylated octapeptide derived from mouse Spry2 inhibits Grb2 from binding FRS2, Shp2 or mouse Spry2 in vitro and blocks activation of the extracellular-signal-regulated kinase (ERK) in cells stimulated by growth factor. A non-phosphorylated Spry mutant cannot bind Grb2 and acts as a dominant negative, inducing prolonged activation of ERK in response to FGF and promoting the FGF-induced outgrowth of neurites in PC12 cells. Our findings suggest that Spry functions in a negative feedback mechanism in which its inhibitor activity is controlled rapidly and reversibly by post-translational mechanisms.

Involvement of the p38 mitogen-activated protein kinase pathway in transforming growth factor-beta-induced gene expression.

Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, is suggested to be involved in TGF-β-induced gene expression, but the signaling mechanism from TAK1 to the nucleus remains largely undefined. We have found that p38 mitogen-activated protein kinase, and its direct activator MKK6 are rapidly activated in response to TGF-β. Expression of dominant negative MKK6 or dominant negative TAK1 inhibited the TGF-β-induced transcriptional activation as well as the p38 activation. Constitutive activation of the p38 pathway in the absence of TGF-β induced the transcriptional activation, which was enhanced synergistically by coexpression of Smad2 and Smad4 and was inhibited by expression of the C-terminal truncated, dominant negative Smad4. Furthermore, we have found that activating transcription factor-2 (ATF-2), which is known as a nuclear target of p38, becomes phosphorylated in the N-terminal activation domain in response to TGF-β, that ATF-2 forms a complex with Smad4, and that the complex formation is enhanced by TGF-β. In addition, expression of a nonphosphorylatable form of ATF-2 inhibited the TGF-β-induced transcriptional activation. These results show that the p38 pathway is activated by TGF-β and is involved in the TGF-β-induced transcriptional activation by regulating the Smad-mediated pathway.

JNK functions in the non-canonical Wnt pathway to regulate convergent extension movements in vertebrates

Recent genetic studies in Drosophila identified a novel non‐canonical Wnt pathway, the planar cell polarity (PCP) pathway, that signals via JNK to control epithelial cell polarity in Drosophila. Most recently, a pathway regulating convergent extension movements during gastrulation in vertebrate embryos has been shown to be a vertebrate equivalent of the PCP pathway. However, it is not known whether the JNK pathway functions in this non‐canonical Wnt pathway to regulate convergent extension movements in vertebrates. In addition, it is not known whether JNK is in fact activated by Wnt stimulation. Here we show that Wnt5a is capable of activating JNK in cultured cells, and present evidence that the JNK pathway mediates the action of Wnt5a to regulate convergent extension movements in Xenopus. Our results thus demonstrate that the non‐canonical Wnt/JNK pathway is conserved in both vertebrate and invertebrate and define that JNK has an activity to regulate morphogenetic cell movements.

A novel SAPK/JNK kinase, MKK7, stimulated by TNFalpha and cellular stresses.

Stress‐activated protein kinase (SAPK)/c‐Jun N‐terminal kinase (JNK), a member of the MAP kinase (MAPK) superfamily, is thought to play a key role in a variety of cellular responses. To date, SEK1/MKK4, one of the MAP kinase kinase (MAPKK) family of molecules, is the only SAPK/JNK kinase that has been cloned. Here we have cloned, identified and characterized a novel member of the mammalian MAPKKs, designated MKK7. MKK7 is most similar to the mediator of morphogenesis, hemipterous (hep), in Drosophila. Immunochemical studies have identified MKK7 as one of the major SAPK/JNK‐activating kinases in osmotically shocked cells. While SEK1/MKK4 can activate both the SAPK/JNK and p38 subgroups of the MAPK superfamily, MKK7 is specific for the SAPK/JNK subgroup. MKK7 is activated strongly by tumour necrosis factor α (TNFα) as well as by environmental stresses, whereas SEK1/MKK4 is not activated by TNFα. Column fractionation studies have shown that MKK7 is a major activator for SAPK/JNK in the TNFα‐stimulated pathway. Moreover, we have found that overexpression of MKK7 enhances transcription from an AP‐1‐dependent reporter construct. Thus, MKK7 is an evolutionarily conserved MAPKK isoform which is specific for SAPK/JNK, is involved in AP‐1‐dependent transcription and may be a crucial mediator of TNFα signalling.

Identification of Two Smad4 Proteins in Xenopus THEIR COMMON AND DISTINCT PROPERTIES

Smad family proteins have been identified as mediators of intracellular signal transduction by the transforming growth factor-β (TGF-β) superfamily. Each member of the pathway-restricted, receptor-activated Smad family cooperates and synergizes with Smad4, called co-Smad, to transduce the signals. Only Smad4 has been shown able to function as a common partner of the various pathway-restricted Smads in mammals. Here we have identified a novel Smad4-like molecule in Xenopus (XSmad4β) as well as a Xenopus homolog of a well established Smad4 (XSmad4α). XSmad4β is 70% identical to XSmad4α in amino acid sequence. Both of the Xenopus Smad4s can cooperate with Smad1 and Smad2, the pathway-restricted Smads specific for bone morphogenetic protein and TGF-β, respectively. However, they show distinct properties in terms of their developmental expression patterns, subcellular localizations, and phosphorylation states. Moreover, XSmad4β, but not XSmad4α, has the potent ability to induce ventralization when microinjected into the dorsal marginal region of the 4-cell stage of the embryos. These results suggest that the two XenopusSmad4s have overlapping but distinct functions.

Leucine-rich repeat kinase LRRK1 regulates endosomal trafficking of the EGF receptor

Activation of the epidermal growth factor receptor (EGFR) not only initiates multiple signal-transduction pathways, including the MAP kinase (MAPK) pathway, but also triggers trafficking events that relocalize receptors from the cell surface to intracellular endocytic compartments. In this paper, we demonstrate that leucine-rich repeat kinase LRRK1, which contains a MAPKKK-like kinase domain, forms a complex with activated EGFR through an interaction with Grb2. Subsequently, LRRK1 and epidermal growth factor (EGF) are internalized and co-localized in early endosomes. LRRK1 regulates EGFR transport from early to late endosomes and regulates the motility of EGF-containing early endosomes in a manner dependent on its kinase activity. Furthermore, LRRK1 serves as a scaffold facilitating the interaction of EGFR with the endosomal sorting complex required for transport-0 complex, thus enabling efficient sorting of EGFR to the inner vesicles of multivesicular bodies. Our findings provide the first evidence that a MAPKKK-like protein regulates the endosomal trafficking of EGFR.

Expression of Siamois and Twin in the blastula Chordin/Noggin signaling center is required for brain formation in Xenopus laevis embryos

The blastula Chordin- and Noggin-expressing (BCNE) center located in the dorsal animal region of the Xenopus blastula embryo contains both prospective anterior neuroectoderm and Spemann organizer precursor cells. Here we show that, contrary to previous reports, the canonical Wnt target homeobox genes, Double knockdown of these genes using antisense morpholinos in Xenopus laevis blocked head formation, reduced the expression of the other BCNE center genes, upregulated Bmp4 expression, and nullified hyperdorsalization by lithium chloride. Moreover, gain- and loss-of-function experiments showed that Siamois and Twin expression is repressed by the vegetal transcription factor VegT. We propose that VegT expression causes maternal beta-Catenin signals to restrict Siamois and Twin expression to the BCNE region. A two-step inhibition of BMP signals by Siamois and Twin-- first by transcriptional repression of Bmp4 and then by activation of the expression of the BMP inhibitors Chordin and Noggin--in the BCNE center is required for head formation.

Xenopus FRS2 is involved in early embryogenesis in cooperation with the Src family kinase Laloo

FRS2 has been identified in mammalian cells as a protein that is tyrosine phosphorylated and binds to Grb2 and Shp2 in response to fibroblast growth factor (FGF) or nerve growth factor (NGF) stimulation. But neither its existence in other vertebrate classes or invertebrates nor its function during embryonic development has been defined. Here we have identified and characterized a Xenopus homolog of FRS2 (xFRS2). xFRS2 is tyrosine phosphorylated in early embryos, and overexpression of an unphosphorylatable form of xFRS2 interferes with FGF‐dependent mesoderm formation. The Src family Kinase Laloo, which was shown to function in FGF signaling during early Xenopus development, binds to xFRS2 and promotes tyrosine phosphorylation of xFRS2. Moreover, xFRS2 and Laloo are shown to bind to Xenopus FGF receptor 1. These results suggest that xFRS2 plays an important role in FGF signaling in cooperation with Laloo during embryonic development.

The TGF‐β family member derrière is involved in regulation of the establishment of left–right asymmetry

Although a number of genes that are involved in the establishment of left–right asymmetry have been identified, earlier events in the molecular pathway developing left–right asymmetry remain to be elucidated. Here we present evidence suggesting that the transforming growth factor‐β family member derrière is involved in the development of left–right asymmetry in Xenopus embryos. Ectopic expression of derrière on the right side can fully invert cardiac and visceral left–right orientation and nodal expression, and expression of a dominant‐negative form of derrière on the left side can partially randomize the left–right orientation and nodal expression. Moreover, while expression of the dominant‐negative derrière does not inhibit the activity of Vg1 directly, it can rescue the altered left–right orientation induced by Vg1. Vg1 can induce derrière in animal cap explants. These results suggest that derrière is involved in earlier molecular pathways developing the left–right asymmetry.

EGFR-dependent phosphorylation of leucine-rich repeat kinase LRRK1 is important for proper endosomal trafficking of EGFR

Endocytosis and subsequent delivery of activated EGFR to lysosomes are essential for the termination of EGFR signaling. It is shown that EGFR regulates the kinase activity of LRRK1 via tyrosine phosphorylation and that this is required for proper regulation of endosomal trafficking of EGFR.