Richard Cerione, College of Veterinary Medicine

The Cerione laboratory has been interested in the molecular mechanisms of signal transduction, using a combination of biochemical and genetic approaches, and more recently combining this with structural studies using X ray crystallography. At present, two main areas are being pursued in the Cerione laboratory. The first involves studies of the regulation and structural characterization of a small single-chain GTP-binding protein, Cdc42, that was discovered by members of the laboratory some years ago and has since been shown to play critical roles in cell growth and in the establishment of cell polarity and cytokinesis.

Efforts are being directed toward identifying the cellular regulators and targets of this important GTP-binding protein and in understanding their interactions in molecular detail through X ray crystallography. Particular emphasis has focused on regulators of the GTP-binding/GTP hydrolytic cycle of Cdc42. Three types of regulators have been identified, a guanine nucleotide exchange factor (Dbl) that stimulates the activation of Cdc42 by promoting GTP-GDP exchange and when amino-terminal truncated is capable of inducing malignant transformation, a GTPase-activating protein (GAP) that catalyzes the GTP hydrolytic reaction, and a GDP-dissociation inhibitor (GDI) that blocks the GTP-binding/GTPase cycle by inhibiting both GTP-GDP exchange and GTP hydrolysis.

During the past year, the three dimensional structures for Cdc42 complexed to its GAP in the presence of aluminum fluoride (which acts as a transition-state analog for GTP hydrolysis), and Cdc42 bound to its GDI have been solved by X-ray crystallography. These structures have provided important mechanistic insights into how the GAP stimulates GTP hydrolysis and how the GDI acts as a negative regulator of Cdc42 signaling. These structures have also highlighted points for site-directed mutagenesis that would be expected to yield useful Cdc42 mutants for studying its cellular functions.

The Cerione laboratory has also been engaged in identifying and biochemically characterizing target/effectors for Cdc42. These have included a serine/threonine kinase, PAK (for p21-activated kinase), that appears to send signals to the actin cytoskeleton and to the nucleus, a tyrosine kinase called ACK (for activated-Cdc42 kinase) which appears to play a role in endocytotic processes, and various other putative target/effectors that suggest a possible role for Cdc42 in intracellular trafficking and secretion. Emphasis is being placed on identifying additional binding partners for the different targets which may play roles in faciliating and/or modulating Cdc42 signals and in determining the three dimensional structures for different Cdc42/target complexes.

A second major area in the laboratory is focused on the biochemical and cellular studies of a GTP-binding protein/transglutaminase (TGase) which catalyzes the transamidation of proteins in a GTP-dependent and ligand (retinoic acid, heregulin)-dependent manner. Recently, the laboratory has obtained evidence that the retinoic acid-dependent transamidation of the retinoblastoma (Rb) gene product to a polyamine donor molecule results in the stabilization of Rb against caspace-mediated proteolysis and ensures the stability of this cell-cycle check-point protein during the cell-cycle arrest that accompanies cellular differentiation. Efforts in the laboratory are being directed toward understanding the connection between the GTP-binding/GTP hydrolytic cycle of the TGase and its transamidation activity, how retinoic acid stimulates the activation of the TGase, and determining the three dimensional structure of the TGase in collaboration with the Clardy laboratory.