Signaling Pathways in Development and Cancer

Model for effect of phosphotyrosine (pY)-dependent substrate turnover in regulating Src signaling.

Intercellular communication coordinates cell proliferation, differentiation, migration and death during development and homeostasis. Cells exchange diffusible or contact-dependent signals that are transduced by highly conserved networks of protein and lipid kinases, small GTPases, second messengers, actin regulators, transcription factors and other intracellular signaling molecules. Many of these relay molecules are tightly regulated in expression or activity. If signaling proteins are deregulated by over-expression or mutation, then downstream responses are activated, frequently leading to a transformed malignant phenotype. Correspondingly, the inhibition, deletion or functional inactivation of signal transduction proteins can block cellular responses and inhibit transformation.

For any signaling protein, the key questions are how is it regulated and what does it do? Some signal transduction pathways are linear, with a single input and single output, but others are networks with many inputs and many effectors. Teasing out the activators, substrates, and regulatory loops is much more complex.

One signaling "hub" is the tyrosine kinase Src. Src is activated by dephosphorylation at one site, phosphorylation at another, and by inter- and intra-molecular binding interactions. Src activators include various transmembrane receptor tyrosine kinases, several protein-tyrosine phosphatases, and cell-cell (cadherin) and cell-matrix (integrin) adhesion molecules. Its substrates include a variety of cytoskeletal regulators, cell surface receptors, signaling proteins and transcription factors. Adding to the complexity,

Image from a time lapse movie of neurons migrating in slice culture. Neurons were labeled three days previously by in utero electroporation with a green fluorescent actin marker and a red fluorescent nuclear marker. (Susumu Antoku)

Src has seven close relatives (paralogs) in vertebrates, many of which are co-expressed, leading to functional overlap and partial redundancy. Uncovering Src functions using genetics has been problematic, and pleiotropic phenotypes result when multiple Src kinases are deleted together. It is clear, however, that over-expression of deregulated Src mutants is oncogenic, due to the increased phosphorylation of specific cell proteins.  

We are using two different systems to understand the roles of Src kinases and other signaling molecules in regulating cell migration.

1. The Reelin signaling pathway.
Reelin is a secreted protein that coordinates neuron migrations in various regions of the developing mammalian central nervous system. The use of embryonic stem cell technology to create mutant alleles, coupled with transient in vivo genetic manipulation and imaging and molecular and cellular biology, enables these neuron migrations to be described and analyzed in great detail.

2. Epithelial and fibroblast growth and cell migration in vitro.
While tyrosine phosphorylation is actively reversed by dephosphorylation, proteolysis is also important. Certain phosphotyrosyl-proteins are targeted for polyubiquitylation and proteasome-dependent degradation, providing a second level of negative regulation. Genetic ablation of this second line of defense deregulates cell proliferation and migration and other hallmarks of malignant transformation.