Studying MAP Kinase Pathways During Early Development of Xenopus laevis
The following chapter describes several methods involved in the detection of MAPK activities and phosphorylated proteins during early development of Xenopus laevis. The Xenopus embryo provides a powerful platform for biochemical studies. We describe here
- PDF / 1,731,534 Bytes
- 12 Pages / 504 x 720 pts Page_size
- 57 Downloads / 186 Views
1. Introduction One of the major challenges in developmental biology is to understand how cells integrate multiple signaling pathways to achieve different cell fates. Germ layer specification and patterning of the Xenopus embryo are regulated by secreted signaling proteins. For example, fibroblast growth factor (FGF) induces mesoderm formation at blastula, while dorsal-ventral gradients of Wnt and bone morphogenetic protein (BMP) determine the different cellular fates along the axis at gastrula. One interesting research question is how these signals are transduced and integrated within cells to
Rony Seger (ed.), MAP Kinase Signaling Protocols: Second Edition, Methods in Molecular Biology, vol. 661, DOI 10.1007/978-1-60761-795-2_25, © Springer Science+Business Media, LLC 2010
409
410
Keren and Bengal
specify the embryo axis. Mitogen-activated protein kinases (MAPKs) function at the earliest cell specification and patterning events. To regulate these processes, MAPK together with other signaling molecules converge at the level of transcription factors to fine tune their activities. ERK MAPK is involved in mesoderm specification downstream to FGF signaling (1, 2). The p53 protein is a mediator of MAPK signaling in this process. FGF/MAPK causes the phosphorylation of p53 at serine residues 6 and 9, promoting a robust biochemical interaction of p53 with TGFbactivated Smads. The p53-Smad complex is active in mesoderm induction (3). In the process of axis formation, FGF/MAPK and BMP/Smad1 pathways are integrated. At the ventral side of the embryo, BMPR phosphorylates C-terminal residues of Smad1 to induce its translocation into the nucleus, while at the dorsal side FGF/MAPK phosphorylates residues within the linker region of Smad1 to terminate its transcriptional activity (4, 5). Phosphorylation of the linker region of Smad1 by ERK MAPK serves as a priming event for phosphorylation at adjacent serine residues by GSK3. This additional phosphorylation targets the protein to the ubiquitin degradation system (reviewed in ref. 6). Therefore, dorsal activation of ERK MAPK signaling is an additional strategy of the organizer to terminate BMP activity via Smad1. Similarly, FGF/MAPK signaling is necessary for neuronal induction via the termination of Smad1 activity (4, 5). On the other hand, antagonism of BMP signaling at the dorsal end induces the ERK MAPK pathway (7, 8). Therefore, a loop between BMP and ERK MAPK is established at the dorsal side of the embryo. We have recently shown that p38 MAPK is also highly active in the dorsal organizer and functions via the phosphorylation of the CREB transcription factor in regulating the expression of chordin (9). Therefore, both p38 and ERK MAPK function by different strategies to prevent BMP signaling at the Spemann organizer. The methods for studying MAPK in the Xenopus embryo were adopted from other systems and were modified for Xenopus. The Xenopus embryo has certain advantages and disadvantages with regard to the study of biochemical mechanisms. Among the advantages, one cou
Data Loading...
