Ribbon diagram for the CBC in complex with the RNA cap analog m7GpppG. The three structural domains of CBP80 and the three main regions of CBP20 are shown, together with the m7GpppG molecule (ball and stick). Taken from Calero et al., 2002.
The research efforts of my laboratory have focused on understanding the molecular mechanisms by which signals are transmitted from cell surface receptors to biological effectors. In particular, we have been interested in identifying new signaling molecules that influence the growth and differentiation of mammalian cells. Three areas of research are currently being pursued in the laboratory.
The first involves studies of the regulation and structural characterization of a cell-division-cycle, Ras-related GTP-binding protein, Cdc42. This GTP-binding protein and its regulators appear to play critical roles in cell growth, the establishment of cell polarity, and cytokinesis. In these studies, we are using a variety of biochemical, molecular biology-based, and structural techniques to obtain new insights into how this Ras-like protein, through interactions with a variety of cellular targets, can coordinate cytoskeletal changes with cell cycle progression and cell division.
A second area of research interest focuses on the structure-function comparisons of heterotrimeric G proteins and Ras-like GTP-binding proteins, where we have used the retinal G protein, transducin, as a model for developing novel fluorescence approaches to study G protein activation and G protein-target interactions.
The third area of interest concerns the identification and structure-function characterization of novel GTP-binding activities present in the nucleus and engaged in regulating RNA metabolism and/or cell cycle progression. At present, we are studying two such nuclear activities. One is an 80 kDa dual function GTP-binding protein/transglutaminase which we purified from the nuclear pore and appears to be regulated by retinoic acid under conditions of cellular apoptosis. The second is an 18 kDa RNA cap-binding protein, CBP20, which plays an essential role in the splicing of precursor messenger RNA and in certain aspects of RNA nucleocytoplasmic transport. We have shown that certain growth factor receptor tyrosine kinases can initiate signaling pathways that lead to the nucleus and culminate in the stimulation of the GTP-binding and RNA cap-binding activity of CBP20. We are now trying to delineate the molecular mechanisms underlying the regulation of each of these nuclear GTP-binding activities.
In pursuing each of these areas of research, we are striving to use a combination of biophysical and molecular biology-based approaches to obtain as complete a picture as possible regarding how growth factor receptors and GTP-binding proteins mediate communication between the plasma membrane and the nucleus. In many cases, this has involved developing fluorescence spectroscopic approaches (e.g.,resonance energy transfer, fluorescence anisotropy) to directly monitor protein-protein interactions that comprise different signaling pathways. More recently, X-ray crystallo-graphic analysis is being used to obtain detailed information regarding the protein interfaces that are involved in signaling interactions. We hope to utilize this infor-mation to design new classes of dominant-negative mutant signaling molecules that will serve as powerful reagents for in vivostudies.