These results suggest that PYK2 is an upstream kinase required for VEGF-induced tyrosine phosphorylation of p130Cas

These results suggest that PYK2 is an upstream kinase required for VEGF-induced tyrosine phosphorylation of p130Cas. (p130Cas15F) was adequate to inhibit growth factor-mediated migration of glioma and endothelial cells. These data demonstrate for the first time the importance of the NRP1 intracellular website in mediating a specific signaling pathway downstream of several receptor tyrosine kinases and determine a critical part for a novel NRP1-p130Cas pathway in the rules of chemotaxis. Neuropilin-1 (NRP1) is definitely a coreceptor for vascular Fingolimod endothelial growth element (VEGF) in endothelial cells and is essential for embryonic angiogenesis and vascular development (10, 29). Though the precise cellular functions of NRP1 have yet to be elucidated, there is a growing body of evidence supporting a key part for NRP1 in the migration of both endothelial and tumor cells (9, 11, 15, 19). NRP1 is definitely thought to act as a coreceptor for VEGF by forming complexes with the VEGF receptor tyrosine kinase (RTK) VEGFR2. Complexation between NRP1 and VEGFR2 enhances VEGF binding, and inhibition of complex formation is associated with reduced VEGFR2 phosphorylation, intracellular signaling, mitogenesis, cell migration, and angiogenesis (16, 18, 28, 34, 35). However, the precise part of NRP1 in VEGF signaling remains unclear. Recent evidence shows that NRP1 also regulates tumor and vascular cell functions stimulated by additional growth factors, such as hepatocyte growth element (HGF) and platelet-derived growth element (PDGF). Overexpression of NRP1 promotes tumor progression by potentiating the effect of the HGF/c-Met pathway, and tumor cell invasion mediated from the HGF/c-Met pathway is dependent on NRP1 through an association with c-Met (11, 15). Furthermore, NRP1 and NRP2 can bind HGF and mediate HGF activation of endothelial cell migration and proliferation (30). A recent report showed that NRP1 is also required for tumor cell-derived PDGF-mediated migration of clean muscle mass cells (2). While these results show that NRP1 is required for ideal growth element signaling important for cell motility, it remains unclear whether NRP1 is critical for specific signaling events induced by growth factors and what those important NRP1-mediated signaling events are. The 44-amino-acid intracellular website of NRP1 lacks a defined signaling function but contains the carboxy-terminal consensus PDZ (postsynaptic denseness 95, disk large, zona occludens 1) website binding motif SEA, which associates with the PDZ website protein synectin, also called neuropilin-interacting protein 1 (NIP-1), or RGS-GAIP-interacting protein 1 (GIPC1) (3). The NRP1 intracellular website, through its association with synectin, has been implicated in NRP1-mediated migration, VEGF-mediated vesicular trafficking, and NRP1/VEGFR2 complex formation (20, 25, 34). Furthermore, manifestation of an NRP1 mutant form lacking the C-terminal SEA residues or knockdown of synectin disrupted vessel formation in zebrafish embryos, phenocopying the effects of NRP1 knockdown (33). Recently, we reported that NRP1 is definitely modified by the addition of chondroitin sulfate and that overexpression of a nonmodifiable mutant (S612A) form of NRP1 prospects to improved invasion Fingolimod of U87MG glioma cells, which is dependent within the adapter protein p130Cas (9). Here, we investigated the part of NRP1 in p130Cas signaling in chemotactic reactions to growth factors. We display that NRP1 Fingolimod is essential for tyrosine phosphorylation of p130Cas in response to HGF and PDGF in U87MG glioma cells and VEGF Fingolimod in endothelial cells. In addition, expression of an NRP1 mutant form lacking the intracellular domain name (NRP1C) indicated that this domain name is crucial for NRP1-mediated RTK signaling. Furthermore, knockdown of either NRP1 or p130Cas or expression of NRP1C or a mutant form of p130Cas deficient in all 15 tyrosines of the YXXP motif within the substrate domain name (SD) (p130Cas15F) inhibited the growth factor-mediated migration of glioma and endothelial cells. These results indicate that NRP1 plays a central role TLR4 in growth factor signaling via p130Cas, thus identifying a novel mechanism regulating cell motility. MATERIALS AND METHODS Cell culture. U87MG cells (a kind gift of P. Parker) were cultured in Dulbecco’s altered Eagle’s.

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