Biochemistry: molecular and structural biochemistry of growth factors; cellular biochemistry of transformed cells and oncogenic protein kinases.

Cells have evolved a complex set of controls to regulate their growth. External factors interact with cellular receptors, resulting in activation of a variety of signal transduction pathways. We are studying the biochemical control of cell growth and division at many levels within these various pathways.

The first area of signal transduction we are studying involves the family of fibroblast growth factor receptors (FGFR), particularly FGFR2 and FGFR3. FGFRs are high affinity signaling receptors for fibroblast growth factors (FGFs). Binding of FGFs to these receptors causes dimerization, resulting in autophosphorylation of the kinase domain and subsequent interactions with effector signaling proteins. Point mutations in members of the FGFR family cause a variety of human congenital skeletal disorders, including: achondroplasia, or human dwarfism; thanatophoric dysplasia, a skeletal malformation syndrome fatal at birth; and the craniosynostosis syndromes, such as Crouzon syndrome, Apert syndrome, and Jackson-Weiss syndrome, which cause cranial malformation and in some cases other defects such as syndactyly of the hands or feet. Work in our lab has shown that FGFR3 mutations causing achondroplasia (Gly 380->Arg) and thanatophoric dysplasia type II, TDII (Lys650->Glu), lead to constitutive activation of the receptor. Our data also suggest that the TDII mutation mimics the conformational changes of the tyrosine kinase domain normally initiated by ligand binding and autophosphorylation. We have also examined mutations in FGFR2, associated with Crouzon syndrome, by generating FGFR2/Neu chimeras. These mutant constructs demonstrate that Crouzon mutations are able to signal via a heterologous receptor tyrosine kinase. Our results show that Crouzon syndrome develops from constitutive activation of FGFR2 caused by receptor dimerization, thus increasing downstream signaling. These results have broad implications in understanding the molecular basis of human developmental disorders involving mutations in the FGFR family.

Another area of interest involves signal transduction of the v-mos oncogene and its cellular counterpart mos, a serine/threonine protein kinase, which activates MAP kinase kinase (MEK). Using a yeast two-hybrid system, we have identified a number of novel proteins that interact with mos and cdc2. These proteins are being characterized by examining their effects on maturation promoting factor (MPF) and MEK activation during meiotic maturation, and their potential as cell cycle regulatory proteins in mammalian cells. MPF is composed of two proteins: cdc2 (a serine/threonine kinase) and a B-type cyclin that acts as a regulatory unit for cdc2. This factor was one of the first protein-complexes identified as a regulator of the cell cycle. Activity of MPF during the cell cycle is regulated by cdc2 phosphorylation and association with cyclin. Currently, we are examining the role of phosphorylation in regulating the subcellular localization of cyclin B1. We have identified five Ser residues in cyclin B1 that are phosphorylation sites, four of which map to a recently identified cytoplasmic retention signal (CRS). The CRS appears to be responsible for the cytoplasmic localization of B-type cyclins, although the mechanism is still unclear. Our results demonstrate that phosphorylation within the CRS regulates nuclear translocation of cyclin B1, which is a requirement for biological activity. This work will help increase our understanding of cell cycle control.

Another current focus in the laboratory entails the study of PDGF (platelet-derived-growth-factor) and its interactions with the PDGF receptor. PDGF is a heterodimer of two homologous subunits (the A-chain and the B-chain) known as c-sis, the normal counterpart of the v-sis oncogene. Using protein biochemistry and molecular biology techniques, we have been studying the structure-function relationships of these proteins. Our work has demonstrated that functional ligand/receptor interactions are restricted to certain subcellular compartments, such as the late Golgi complex and/or the plasma membrane. Ligand/receptor interactions in other subcellular compartments (such as the ER or the early Golgi complex) do not lead to functional signal transduction. Current research involves characterization of the cysteine-knot portion of the v-sis oncogene and downstream effectors of the PDGF signaling pathway, such as raf and ras.

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