Dr. Jonathan A. Cooper

Signaling Pathways in Development and Cancer

We are interested in the in vivo functions and mechanisms of action of adaptor proteins related to Drosophila Disabled, and in the signaling pathways used by Src, Fyn and other Src-family tyrosine kinases to regulate normal cell behavior and malignant transformation. We study these functions in cultured vertebrate cells and in genetically-engineered mice.

1. Disabled 1, Src family kinases and brain development

Disabled adaptor proteins contain a highly conserved PTB domain which binds to phosphoinositides (PIP2) and proteins that contain a FXNPXY sequence. Our studies in worms and mice show that Disabled proteins interact with cell surface receptors, and either regulate their subcellular localization or relay downstream signals. While not all interacting proteins have been identified, all Disabled proteins we have studied bind to transmembrane receptors related to the Low Density Lipoprotein (LDL) receptor. These receptors act as import carriers for various signaling molecules and nutrients, and some have signaling roles.

We have found that Disabled 1 (Dab1) is essential for normal neuron migrations during mammalian brain development. Two lipoprotein receptors expressed on CNS neurons have partially redundant roles in binding a signaling molecule called Reelin. Clustering of these receptors by Reelin induces the tyrosine phosphorylation of Dab1 in the neurons. Dab1 tyrosine phosphorylation, by Src and its close relative Fyn, is essential for proper migration of post-mitotic neurons before they undergo terminal differentiation. In mice that are mutant for Dab1, or for both Src and Fyn, the neurons differentiate in incorrect locations within the developing brain. This pathway also regulates dendrite development and synaptic plasticity later in life. We are characterizing the pathways that link Dab1, Src and Fyn to the migration machinery. One pathway involves PI3 kinase and Akt. Another involves the Crk and CrkL adaptor proteins and tyrosine phosphorylation of the C3G Rap1 guanine nucleotide exchange factor. We have evidence that Dab1 has two important functions: one is to activate Src and Fyn and the other is to act as a scaffold to assemble signaling complexes. Both functions are needed for normal development. We are also studying the mechanism that turns off signaling, and discovered a unique phenotype when pathway inactivation is prevented in vivo. We are using neuron cultures, in utero electroporation of DNA into developing fetal brain, and genetically-engineered mice to dissect the signaling mechanism and underlying cell biology further.

2. Disabled 2 and cellular transport

Disabled 2 (Dab2) is encoded by a gene that is down-regulated in many human cancers. Re-expression of Dab2 in cancer cell lines has been shown to cause apoptosis and slow cell proliferation. The mechanism of this is unknown. So far, the only in vivo function we can ascribe to Db2 is the endocytosis of lipoprotein receptors. Lipoprotein receptors frequently act as co-receptors or endocytic receptors for signaling molecules. For example, Megalin binds Sonic Hedgehog and BMP4. We found that Dab2 conditional knockout inhibits gastrulation and decreases endocytosis of Megalin in specific cell types. Dab2 is required in the visceral endoderm of the pregastrula embryo, later in the visceral yolk sac, and in the adult kidney proximal tubule for endocytosis of Megalin. Uptake of the LDL receptor in fibroblasts requires Dab2 if another adaptor, ARH is absent. Dab2 functions by binding to cell membranes, receptors, clathrin and accessory proteins that help form a clathrin-coated pit. We are currently screening for other proteins whose endocytosis requires Dab2. Changes in cell surface receptors may be responsible for the changes in Dab2 expression in cancer cells.

3. Src family kinases

In addition to uncovering the mechanism of Src and Fyn in regulating neuron migrations, we are also interested in the role of Src activation in cancer cells. Many human tumors have increased Src kinase activity. However, activating mutations in the Src gene, such as are found in Rous sarcoma virus, are exceedingly rare in human cancers. Indeed, activating mutations in any of the eight Src family kinase genes are virtually unknown in human samples. It seems likely that such mutations have both positive and negative effects on malignant transformation in vivo. We are developing mouse models to study the role of activating mutations in Src family kinases in cancer and development