Protein tyrosine phosphorylation is an important event in the control of normal cellular processes such as proliferation, differentiation, migration and early patterning events in the vertebrate embryo. The activation of growth factor receptor and non-receptor tyrosine kinase (PTKs) initiates many of these events. Evidence also indicates that when normal PTK function is perturbed during embryogenesis, major phenotypic changes or defects can occur. The extent to which a protein is phosphorylated in vivo may be thought of as a balance between the opposing activities of the PTKs and the protein tyrosine phosphatases (PTPs). The PTPs may act directly on PTK signaling pathways either by a dephosphorylation event necessary for activation, or may act by dephosphorylating signaling molecules and thus attenuating the signaling mechanism. Both of these processes may come into play during growth and development, but their role is poorly understood. The long term goal of this project is to characterize the molecular mechanisms that regulate tyrosine kinase signaling pathways during growth migration and development. This proposal will focus on the role of Xenopus PTPalpha since it is widely expressed both during development and in adult tissues. Evidence from other experimental systems indicates that PTPalpha may be an important regulator of tyrosine kinase signaling, possibly by regulating pp60(c-src) tyrosine kinase activity. We have recently identified additional substrates for PTPalpha including possible interaction with components of the fibroblast growth factor receptor (FGFR) signaling pathway.
The specific aims of the proposal are: 1) To determine the biological consequences of overexpression of wild-type and mutant forms of XPTPalpha in oocytes and developing Xenopus embryos. The approach will be to microinject synthetic mRNAs encoding wild-type or mutant forms of XPTPalpha and to analyze for embryonic phenotypes, and changes in embyronic stage- or tissue-specific gene expression. In addition, oocytes and embryos will be examined for changes in the phosphorylation state of specific polypeptides. 2) To identify and characterize physiologically relevant substrates for PTPalpha. The approach will not only include the identification of XPTPalpha substrates, but the analysis of functional domains of XPTPalpha that mediate interactions with substrates. 3) To identify and characterize mechanisms that regulate the activity of XPTPalpha during oocyte maturation and embryonic development. The approach will concentrate on post-translational mechanisms of control including, in particular, an analysis of the phosphorylation state of PTPalpha at various stages of oocyte and embryonic development. The approaches outlined above will elucidate some of the mechanism by which PTPalpha regulates tyrosine kinase signaling during early development. Because of the nearly ubiquitous distribution of PTPalpha in vertebrates and its apparent regulation of pp60(c-src) and FGFR pathways, these studies are likely to have impact on other areas of biology including cell cycle control, migration and angiogenesis.
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