Cell Migration: Directed cell movement, or chemotaxis, is exhibited during wound healing, angiogenesis, embryonic development, immune function, and during cancer cell metastasis. This dynamic process is highly conserved, as prokaryotes and eukaryotes from Dictyostelium discoideum to human leukocytes exhibit the ability to sense and move in the direction of a chemoattractant (see movie 1 below). The basic steps of cell polarization and chemotaxis are illustrated in the schematic: (Step 1) Nonmotile cells are attached to the underlying extracellular matrix (ECM), most likely through integrin receptors on the cell surface (squares). (Step 2) Cells are exposed to a soluble gradient of growth factor or chemokine, which binds to and activates cell surface receptors and downstream signals. Recent evidence indicates that when eukaryotic cells encounter a chemoattractant gradient they respond by local activation and amplification of signaling events on the side facing the gradient. (Step 3) Although it is not well defined, these signals presumably facilitate localized actin polymerization leading to membrane protrusion in the direction of the gradient. Importantly, the membrane protrusion of a pseudopodium (or lamellipodium) is independent of actual cell body translocation or chemotaxis. The establishment of a dominant leading pseudopodium and rear cell body compartment marks the first sign of morphological polarity. Interestingly, the initial protrusion of a pseudopodium at the cell surface is independent of integrins and the ECM. However, integrins tether the extending membrane to the substratum, which supports sustained and directional pseudopodia growth. Indeed, pseudopodia that do not attach to the ECM rapidly retract back into the cell body. This suggests that new integrin ligation events at the leading front of the extending membrane provide necessary signals to fine-tune and maintain directional growth, while suppressing retraction mechanisms. (Step 4) Once a dominant pseudopodium is formed, cell movement commences in the direction of the gradient as the cell undergoes repeated cycles of membrane extension at the front and retraction of the rear compartment.

To understand the spatiotemporal organization of signaling pathways that mediate morphological polarity of migrating cells, we have developed a unique pseudopodial purification system that facilitates biochemical analysis of this structure (see figure 1 below). Using the pseudopodial purification system, vitro assays of cell migration, and time-lapse confocal microscopy, we are investigating in detail how the canonical Ras/ERK and p130CAS/Crk-II/Rac signaling pathways contribute to directional cell movement (fig 3). Also, our proteomic analysis of the pseudopodial proteome has uncovered several novel proteins important for cell migration including Lasp-1 (fig 2 and PEAK1 (fig 4). We are currently investigating their role in regulating the actin-myosin cytoskeleton during cell migration and cancer cell metastasis.

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).

Figure 2. Time-lapse movie of HepG2 adenocarcinoma cells migrating in response to an insulin chemokine gradient diffusing from a glass micropipette. Note the ability of the cells to sense and move directionally toward the chemoattractant as the pipette is repositioned in the field.

Figure 3. Schematic showing the major compoments of the src/p130CAS/Crk/Rac signaling pathway that control pseudopodial formation and cell movement. Integrin ligation and/or growth factor receptor activation promotes src activation, which phosphorylates at least ten (Y238-Y414) tyrosine residues (YxxP)15 present in the substrate domain of CAS (J. Cell Biol. 140:961, 1998. Biochimica et Biophy. Acta 1692:63, 2004). This in turn provides SH2 binding sites for the Crk adaptor protein and its associated effector and Rac GTPase activating protein DOCK180. The formation of this signaling module at sites of cell adhesion and in the invadopodium (pseudopodium) serve as a molecular switch that drives actin-mediated membrane protrusion leading to increased movement and survival as cells invade the ECM. Importantly, cells deficient in CAS or Crk can not be transformed by v-src and display impaired cell movement and invasion of reconstituted ECM proteins. Abl tyrosine kinase is also activated by integrin and growth factor signaling and serves as a negative feedback loop to control CAS/Crk coupling in invasive cells (J. Biol. Chem. 276:16185, 2001. Oncogene 22:6071, 2003). In this case, Abl phosphorylates Y221 of Crk, which facilitates intramolecular folding of the molecule so that its own SH2 domain interacts with Y221 preventing it’s binding to CAS. PEAK1 is a novel tyrosine kinase that is tyrosine phosphorylated downstream of integrin and growth factor receptor activation. The Src phosphorylation sites reside at Y665 and Y1188 and the Crk SH3 polyproline binding domain is at P1153. Functionally, this protein localizes to the actin cytoskeleton of extending invadopodia, is necessary for proper invadopodia formation and cell movement, is upregulated in highly metastatic cells, and is amplified in greater than 70% of human colon cancers (manuscript in preparation). This new protein is highly significant as it provides a possible biomarker for metastatic cancer and a drugable target for therapeutic intervention of this signaling pathway. Future work will determine how PEAK1 works in conjunction with src/CAS/Crk/Rac signaling to mediate pancreatic cancer progression.

Figure 4. 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 5. 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).