Dr. Jonathan A. Cooper

Cellular and developmental biology of Disabled adaptor proteins and Src-related tyrosine kinases

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 family of non-receptor tyrosine kinases to regulate normal cell behavior and malignant transformation. We study these functions in cultured vertebrate cells and in genetically-engineered mice.

Disabled adaptor proteins:
Disabled adaptor proteins are found in flies, worms and vertebrates, and contain a highly conserved PTB domain which binds to non-phosphorylated tyrosine-containing sequences and to phosphoinositides. While the function of Disabled in flies remains a mystery, 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.

Disabled 1:
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 Disabled 1 in the neurons. Disabled 1 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 Disabled 1, or for both Src and Fyn, the neurons differentiate in incorrect locations within the developing brain. However, many axonal connections are correctly made and mice with these brain defects can live, albeit with coordination defects correlating with an under-developed cerebellum. We are characterizing the signal transduction pathways that relay the signals from Disabled 1, 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 are using neuron cultures, in utero electroporation of DNA into developing fetal brain, and knock-in and conditional mutant mice to understand this pathway further.

Disabled 2:
Disabled 2 is encoded by a gene whose expression is inhibited in many human carcinoma samples and by oncogenes in cell culture. Re-expression of Disabled 2 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 Disabled 2 is that it binds directly to lipoprotein receptors, phospholipids and clathrin to mediate endocytosis. Disabled 2 conditional knockout and knock-in mice have various developmental and physiological defects that seem to be caused by decreased endocytosis of lipoprotein receptors in specific cell types. For example, knockout of Disabled 2 in the visceral endoderm of the pregastrula embryo, in the visceral yolk sac, and in the kidney proximal tubule inhibits endocytosis of megalin and compounds imported by megalin. In other cell types, however, Disabled 2 is partially redundant with other endocytic adaptor proteins. We are currently screening for other proteins whose endocytosis requires Disabled 2. Changes in cell surface receptors may be responsible for the altered biology of Dab2-negative cancer cells.

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.