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Department of
Biochemistry Ph.D. at EMBL and University of Heidelberg, Germany , 1983 1980-1983 European Molecular Biology Laboratory (EMBL) Predoctoral Fellow;
Research: Here is a link to download this document in Adobe Acrobat .PDF format Cancer and Diabetes: Targeting Defects in the Cellular Signaling Circuitry by Molecular Cell Biology, Functional Genomics/Bioinformatics and Proteomics. Goals: A key goal in biomedicine is the development of new therapies for diseases. Cancer and Diabetes rank highly on a global scale. Aspects of both diseases result from alterations in the cellular signaling circuitry that is critical to coordinate the normal cellular processes within one cell and between individual cells in tissues and organs (6,7). The research focus of this team is to unravel the wiring of key signaling circuits and the underlying networks and molecular mechanisms (4,5) that play a role in these diseases as well as in normal cell regulation. We are defining key mechanisms of the same fundamental circuitry that regulates diverse cellular processes in cell proliferation, survival and malignant transformation, cell migration, invasion, wound healing, and metabolism in diverse environments and organs from the liver to the central nervous system as well as in developmental programs. Discovery: A few years ago my colleagues and I co-discovered two families of signaling mediators (Figure 1) in a proteomics screen with the yeast two-hybrid system (11,12). As anticipated in our screen both mediator families, represented by Grb10 (1,2) and PSM/SH2-B (8), emerged as key components of cellular signaling circuits in the control of cell proliferation (9,10) and in the metabolic response to insulin (3), two major areas of our research interest. When we disrupted the function of the mediators in mammalian cells we were intrigued to discover that this triggered a complete block of cell proliferation including malignant cell transformation or of insulin action (Figure 2). In contrast, the same mechanisms were stimulated when we increased mediator levels. These experiments (3,8,9,10) demonstrated the essential role of both mediators in the respective signaling networks that is also supported by gene disruption experiments in transgenic mice. Our results support the potential value of both mediator families as new targets for future clinical therapies to restore the normal cellular wiring of the signaling circuitry. Normal insulin or mitogenic signaling are impaired in Type 2 Diabetes or some types of Cancer, respectively. Strategy: As a critical experimental strategy we are routinely utilizing domain-specific and cell membrane-permeable (Trojan) peptides representing the mediators in a proteomic version of a gene knockout (3). The advantage of this approach over conventional gene knockout strategies lies in its specificity for one functional domain of the mediator that allows the mapping of the specific function of each protein domain individually (Figures 1, 2). A second advantage lies in the rapid applicability to any cell line or tissue including whole animals. Once domain-specific peptides have been designed by routine recombinant DNA procedures and expressed in E. coli they can be introduced into any cell in minutes without further preparation as well as into animal models such as mice where they will spread throughout the whole body.
Normal (or wild-type) mouse fibroblast cells show an organized cytoskeleton of parallel actin stress fibers (WT). The actin cytoskeleton becomes disorganized after the cells have been transformed into metastatic cancer cells (Control, M). We have developed anti-cancer strategies by introducing dominant-negative peptides of the signaling mediator PSM/SH2-B into cells (PSM). This reverts the disorganized cancer cytoskeleton (Control, M) to a largely normal cytoskeleton (PSM, M). Control peptides (Control, M) or peptides of the other signaling mediator (Grb10, M) do not change the disorganized cytoskeleton. The results suggest that the anti-cancer effect of the PSM peptides is specific and may ultimately lead to new therapeutic strategies. Projects: Aided by this strategy we are focusing on new wiring concepts in the Grb10 and PSM/SH2-B-mediated signaling circuitry involving multiple use of the same domain and intra- and inter-molecular interactions resulting in adapter multimers. These concepts are now under intense investigation and experiments help define the potential of cell membrane-permeable peptides as therapeutic drugs. We are particularly interested in applying our tools to the dissection of the signaling complexes and wiring networks formed by the tyrosine kinase receptors of growth factors (15) and peptide hormones (16) including the insulin receptor (Figure 3). We employ proteomics strategies including yeast two-hybrid interactions, in vitro protein-protein affinity binding and co-immunoprecipitiation combined with peptide mass fingerprinting to dissect the individual protein-protein interactions and establish the wiring diagram of the signaling complex (Figure 3). With these strategies we have already identified new wiring partners of Grb10 and PSM/SH2-B. We have designed about a dozen domain-specific peptides (Figure 1) of either mediator that are being used to map their interactions with their protein partners in interaction assays in vitro as well as in functional assays in vivo (Figure 2). With these reagents we have identified roles of the mediators in malignant cell transformation, normal cell proliferation and survival, cell migration, metabolic insulin action, and potentially directly in the cell nucleus in gene transcription (3,8,9,10). Additional roles have been found in development and in the central nervous system (2).
Opportunities: In all described areas projects are available for graduate students and postdoctoral associates that can be tailored to the individual's research interest. Opportunities include to define the molecular details of the signaling circuits and the interactions that we have identified as described above. Since our basic studies are well advanced publishable results should be obtainable rapidly. Projects are available to characterize signaling mediators and their molecular mechanism of action by DNA sequence analysis, cDNA expression, site-directed mutagenesis, biochemical analysis, by physiologic studies in cell culture models and in whole animals (8,13,14). Experimental strategies will capitalize on bioinformatics and functional genomics including combinatorial approaches such as phage display. We are exploiting proteomics strategies (Figure 1) combining cell membrane-permeable (Trojan) peptides and biochemical interaction analysis with peptide mass fingerprinting to dissect the involved signaling complexes (Figure 3). We are evaluating specific physiologic responses by dominant-negative and over-expression strategies (Figure 2). Specific questions will address why and how cancer resulting from specific oncogenes is critically dependent on the function of the adapters, how the adapters interact with other mediators in mitogenic and metabolic signaling, and how they associate with and regulate key signaling mediators such as PI3-kinase in novel modes of interaction, in particular in insulin action.
The underlying mechanisms are of broad significance and shared by the key signaling circuitry that controls development, differentiation, growth and metabolism in most animals. Our research can be applied to efforts in the biotechnology and pharmaceutical industries to design specific molecular therapies or diagnostics. Our work has resulted in patents and licensing fees from the biotechnology industry, about a hundred research papers (see list of 16 selected articles) and has been continuously supported by twenty major research grants and fellowships from government and private sources and foundations. Former trainees in the program have secured positions in academia including independent academic faculty positions and in industry including responsibility for large laboratories. Successful applicants can expect to obtain rigorous training in modern molecular genetics, cell biology, bioinformatics, and proteomics, to significantly expand their publication list, and to develop the skills and the expertise needed to succeed in the biotechnology industry or in a career towards an independent academic position. References:
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Figure 1. Grb10 Family of Alternative Splice Variants (1). Amino acid sequences of mouse Grb10 variants alpha (α), delta (δ), eta (η), and human Grb10 variants beta (β), gamma (γ), epsilon (ε), zeta (ζ) have been aligned for maximum similarity and Pro-rich (PRO), Ras-associating-like (RA), pleckstrin homology (PH), BPS (between the PH and SH2 domains), and Src homology 2 (SH2) domains (including Nedd4 binding motif pSP) have been indicated. Amino acids shown underneath each domain represent a Grb10 sequence motif predicted to be critical in the association with a target domain (identified by type) of independent signaling mediators shown below. Intermolecular interactions are represented by four pairs of dots. Human Grb10 gamma (γ) and mouse Grb10 eta (η) which share the most simple domain structure have been used as a reference and for other variants only changes in the domain structure have been shown. Boxes represent amino acid sequences as indicated by numbers. The unique N-terminal sequences of hGrb10ε is represented by a striped box. See also http://cbr-rbc.nrc-cnrc.gc.ca/thomaslab/grb7.html for more information about the super family. 
Figure 2. PSM SH2 Domain Interferes with Transformed Phenotype (9). 
Figure 3. Wiring Diagram of Grb10 Signaling Complex in Response to Insulin Receptor or Insulin-like Growth Factor-I Receptor (IR/IGF-IR) Activation (1). Grb10 is displayed as an octagon in the center and associated cellular partners are represented by mostly rectangular objects, identified by specific name in white letters. Contact between objects represents a postulated direct cellular association. The mediator Sos is shown as a sickle-shaped object connecting mediators Grb2 and Shc to the distant Ras. The plasma membrane is represented by dual horizontal sheets. Signaling pathways are shown by arrows with physiologic endpoints identified by black letters. Relative sizes and shapes are arbitrary and have been chosen exclusively to optimize the display of putative interactions in two dimensions. 
Rewards: