Directed cell migration, or chemotaxis, in response to a chemokine gradient is involved in development, immune function, inflammation, and cancer cell metastasis. However, little is known about the molecular signaling mechanisms that cause a stationary cell to become directionally motile and metastatic. Ultimately, these signals must target the actin-myosin cytoskeleton of the cell to induce an asymmetrical polarized morphology with a leading pseudopodium (invadapodium) and a rear compartment. Pseudopodia growth is associated with actin polymerization, membrane ruffling, and formation of new focal adhesions and occurs independently from cell body translocation. Establishment of the rear component occurs after pseudopodium formation and is characterized by loss of focal adhesions and strong actin-myosin contraction. Although it is clear that these events are important for cell movement, it has been technically difficult to study the spatial signals that direct the front and rear compartments of polarized cells using large-scale biochemical methods. To address this limitation, our laboratory developed a novel method for the purification of the pseudopodia and rear compartments of cells polarized towards a chemoattractant gradient. Using this novel purification method and phosphotyrosine affinity purification combined with LC-MS/MS protein identification, we discovered a new tyrosine kinase (KIAA2002) which is necessary for cell spreading, pseudopodia formation and proper cell migration. The KIAA2002 protein is highly enriched in the pseudopodium and localizes to the tips of pseudopodial extensions, where it associates with actin-rich membrane ruffles. Integrin-mediated cell adhesion and exposure to cells to growth factors and chemoattractants (LPA, EGF) facilitate tyrosine phosphorylation of KIAA2002. Based on our preliminary data and informatics, KIAA2002 is predicated to be phosphorylated by src kinase and to be a member of the canonical integrin signaling pathway Src/CAS/Crk/DOCK180/Rac-actin. Interestingly, this protein is also amplified in highly metastatic cancer cells including 70% of colon cancer patients with metastatic disease. Structure-function studies outlined in aim 1 will determine the mechanism(s) of how KIAA2002 couples to the migration machinery.
In aim 2 we will determine the role of KIAA2002 in mediating human cancer cell metastasis in vivo.
In this proposal, we will determine how the novel tyrosine kinase KIAA2002 mediates pseudopodia formation and cell migration. We will also determine whether KIAA2002 contributes to cancer progression by regulating cell invasion and metastasis.
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