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.

Recent genetic studies in Drosophila identified a novel noncanonical 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.

Transforming growth factor-␤ (TGF-␤)-activated kinase 1 (TAK1), a member of the mitogen-activated protein kinase kinase kinase family, is suggested to be involved in TGF-␤-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-␤. Expression of dominant negative MKK6 or dominant negative TAK1 inhibited the TGF-␤-induced transcriptional activation as well as the p38 activation. Constitutive activation of the p38 pathway in the absence of TGF-␤ 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-␤, that ATF-2 forms a complex with Smad4, and that the complex formation is enhanced by TGF-␤. In addition, expression of a nonphosphorylatable form of ATF-2 inhibited the TGF-␤-induced transcriptional activation. These results show that the p38 pathway is activated by TGF-␤ and is involved in the TGF-␤-induced transcriptional activation by regulating the Smad-mediated pathway.

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.

Src homology 2-containing phosphotyrosine phosphatase (Shp2) functions as a positive effector in receptor tyrosine kinase (RTK) signaling immediately proximal to activated receptors. However, neither its physiological substrate(s) nor its mechanism of action in RTK signaling has been defined. In this study, we demonstrate that Sprouty (Spry) is a possible target of Shp2. Spry acts as a conserved inhibitor of RTK signaling, and tyrosine phosphorylation of Spry is indispensable for its inhibitory activity. Shp2 was able to dephosphorylate fibroblast growth factor receptor-induced phosphotyrosines on Spry both in vivo and in vitro. Shp2-mediated dephosphorylation of Spry resulted in dissociation of Spry from Grb2. Furthermore, Shp2 could reverse the inhibitory effect of Spry on FGF-induced neurite outgrowth and MAP kinase activation. These findings suggest that Shp2 acts as a positive regulator in RTK signaling by dephosphorylating and inactivating Spry.Receptor tyrosine kinase signaling regulates a wide variety of biological processes in response to extracellular signals, including cellular growth, differentiation and metabolism (1-4). For example, fibroblast growth factor (FGF) 1 stimulates the receptor tyrosine kinase activity of the FGF receptor (FGFR), leading to tyrosine phosphorylation of the docking protein FRS2, consequent recruitment of multiple Grb2-Sos complexes, and activation of the Ras-mitogen-activated protein (MAP) kinase signaling pathway (5-7). Precise control of MAP kinase activation is critical for such cellular responses, and several molecules have been identified that serve to regulate the activation of MAP kinase either positively or negatively.Shp2 is a widely expressed protein-tyrosine phosphatase (PTPase) that seems to play a positive role in the activation of MAP kinase in response to growth factors (8 -14). In response to growth factor stimulation, Shp2 becomes tyrosine-phosphorylated and binds to Grb2, where it seems to act as an adaptor protein to recruit the Grb2-Sos complex to the plasma membrane. This, in turn, leads to Ras activation (6,9,(15)(16)(17). However, several studies have shown that mutation of Shp2 in the putative Grb2 binding site did not interfere with the function of Shp2 in mediating MAP kinase activation (12,18). In contrast, several lines of evidence indicate that the phosphatase catalytic activity of Shp2 is required for MAP kinase activation by FGF and other growth factors (12). Thus, two critical tasks are identifying the substrate for Shp2 and determining how dephosphorylation contributes to the activation of MAP kinase. Recently, it has been reported that a major binding protein of Shp2 is the multi-site docking protein Gab1 (Grb2-associated binder 1) and that this association is required for MAP kinase activation by several growth factors (19 -22). Interestingly, genetic analysis of Corkscrew (Csw), the Drosophila homologue of Shp2, has led to the identification of the Daughter of Sevenless (Dos) protein as a putative Csw substrate (23-25). The Dos protein...