Proteomics: Cell migration requires morphological polarization characterized by formation of a leading pseudopodium (PD) at the front and a trailing rear at the back. This process is controlled by the spatial and temporal organization of protein signaling networks that partition to the front or back of the cell. Reversible protein phosphorylation on serine, threonine, or tyrosine amino acid residues is a major mechanism utilized by migratory cells to coordinate the localization, activation, stability, and assembly of macromolecular signaling complexes. However, aberrant regulation of protein localization and protein phosphorylation in cancer cells contributes to the processes of cell invasion, migration and metastasis. Using unique pseudopodial purification methods developed in the Klemke Lab, we have applied global proteome profiling in combination with newly developed quantitative phosphoproteomics and bioinformatics approaches for comparative analysis of the cell rear (cell body, CB) and PD proteomes of migratory cells. The spatial relationship of greater than 4000 proteins and more than 200 distinct sites of phosphorylation were mapped revealing networks of signaling proteins that partition to the PD and/or the CB compartments. The major networks represented in the PD include integrin signaling, actin-regulatory proteins, and axonal pathfinding, whereas the CB consists of DNA/RNA metabolism, cell cycle regulation and structural maintenance. Our work has provided insight into the spatial organization of signaling networks that control cell movement and provide a comprehensive system-wide profile of proteins and their sites of phosphorylation that control cell polarization. Our work has also led to the discovery of important new proteins and phosphoproteins important for cell migration including the actin binding protein Lasp-1 (J Cell Biol. 2004 May 10; 165(3):421-32.) and the atypical tyrosine kinase PEAK1 (pseudopodial-enriched a typical kinase one). We are currently determining the functional role of these novel signaling molecules and protein networks using standard siRNA gene knockdown technology, molecular biology and confocal microscopy techniques.

Figure 1. Schematic showing microporous filter system for pseudopodial purification and quantitative proteomics and phosphoproteomics using mass spectrometry (MS) (J. Cell Biol. 156:725, 2002. J. Biol. Chem. 278:13016, 2003, PNAS in press, 2007). (Link to larger picture)

Figure 2. Schematic showing the protein interactome of proteins that are enriched (>1.5) in the pseudopodium. Important proteins (integrins, Lasp-1, EGFR) and protein networks (Arp2/3) that control cell migration are shown in circles. Color scale in red shows the relative protein abundance for each protein in the interactome. Solid lines indicate direct protein interactions. Dashed lines indicate functional interactions. Ingenuity Software (http://www.ingenuity.com/) was used to generate the interactome from greater than a thousand pseudopodial proteins.

Figure 3. Confocal micrograph of an NIH 3T3 cell expressing GFP-Lasp-1 (green) and stained with anti-vinculin antibodies to mark focal adhesions (red). Yellow indicates where vinculin and Lasp-1 co-localize in the cell. Lasp-1 is an actin binding protein that is enriched in the leading front of migrating cells and in focal adhesions that reside at the membrane edge of the extending pseudopodium. This protein was identified using the pseudopodial purification and mass spectrometry technologies and shown to be a substrate of Abl and Bcr-Abl tyrosine kinases. It is necessary for proper cell migration and survival (J. Cell Biol. 165:421, 2004). Arrow indicates direction of cell movement.

Figure 4. Confocal micrograph of a Cos-7 cell expressing GFP-PEAK1 (green) and stained with rodamine-phallodin to visualize the F-actin cytoskeleton. PEAK1 (pseudopodial-enriched atypical kinase) is a new tyrosine kinase that operates downstream of integrin and growthfactor receptors to mediate cell migration. PEAK1 is tyrosine phosphorylated and associates with the F-Actin cytoskeleton in membrane ruffles and at the edge of extending pseudopodia. This protein was identified using the pseudopodial purification and mass spectrometry technologies and is necessary for cell migration. We are currently investigating the role of PEAK1 in mediating cell migration and human cancer progression (manuscript in preparation).

Faculty 1000 Review