sq-23377 and lysophosphatidic-acid

Calcium-mediated proteolysis plays an important role in cell migration. Lysophosphatidic acid (LPA), a lipid mediator present in serum, enhances migration of carcinoma cells. The effects of LPA on calpain-mediated proteolysis were, therefore, examined in PC-3, a human prostate cancer cell line.. Cultured PC-3 cells were used in studies utilizing pharmacologic interventions, immunoblotting, and confocal immunolocalization.. Focal adhesion kinase (FAK), a tyrosine kinase involved in cell adhesion, is rapidly proteolyzed in serum-starved PC-3 cells exposed to the calcium ionophore, ionomycin; Nck, p130CAS, PKCα, and Ras-GAP are also degraded. Thapsigargin, which causes more moderate increases in intracellular calcium, induces partial proteolysis of these proteins. Calpain inhibitors block the proteolytic responses to ionomycin and thapsigargin. Ionomycin does not induce proteolysis in cells maintained in serum, suggesting a protective role for growth factors contained in serum. LPA causes minor FAK proteolysis when added alone, but protects against ionomycin-induced proteolysis in a time-dependent manner. LPA also protects against the cell detachment that eventually follows ionomycin treatment. The response to LPA is blocked by an LPA receptor antagonist. A similar effect of LPA is observed in ionomycin-treated Rat-1 fibroblasts. In PC-3 cells, the protective effects of LPA and serum are correlated with phosphorylation and redistribution of paxillin, suggesting roles for phosphorylation-mediated protein-protein interactions.. The complex effects of LPA on calpain-mediated proteolysis of FAK and other adhesion proteins are likely to play a role in the ability of LPA to promote attachment, migration, and survival of prostate cancer cells.

Topics: Adenocarcinoma; Animals; Calpain; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Survival; Drug Screening Assays, Antitumor; Fibroblasts; Focal Adhesion Protein-Tyrosine Kinases; Humans; Ionomycin; Isoxazoles; Lysophospholipids; Male; Paxillin; Phosphorylation; Propionates; Prostatic Neoplasms; Proteolysis; Rats; Thapsigargin

RasGRF family guanine nucleotide exchange factors (GEFs) promote guanosine diphosphate (GDP)/guanosine triphosphate (GTP) exchange on several Ras GTPases, including H-Ras and TC21. Although the mechanisms controlling RasGRF function as an H-Ras exchange factor are relatively well characterized, little is known about how TC21 activation is regulated. Here, we have studied the structural and spatial requirements involved in RasGRF 1/2 exchange activity on TC21. We show that RasGRF GEFs can activate TC21 in all of its sublocalizations except at the Golgi complex. We also demonstrate that TC21 susceptibility to activation by RasGRF GEFs depends on its posttranslational modifications: farnesylated TC21 can be activated by both RasGRF1 and RasGRF2, whereas geranylgeranylated TC21 is unresponsive to RasGRF2. Importantly, we show that RasGRF GEFs ability to catalyze exchange on farnesylated TC21 resides in its pleckstrin homology 1 domain, by a mechanism independent of localization and of its ability to associate to membranes. Finally, our data indicate that Cdc42-GDP can inhibit TC21 activation by RasGRF GEFs, demonstrating that Cdc42 negatively affects the functions of RasGRF GEFs irrespective of the GTPase being targeted.

Topics: Animals; cdc42 GTP-Binding Protein; Chlorocebus aethiops; COS Cells; Enzyme Activation; Guanosine Diphosphate; HeLa Cells; Humans; Ionomycin; Lysophospholipids; Membrane Proteins; Monomeric GTP-Binding Proteins; Organelles; Prenylation; Protein Interaction Mapping; Protein Processing, Post-Translational; Protein Structure, Tertiary; ras Guanine Nucleotide Exchange Factors; ras-GRF1; Recombinant Fusion Proteins; RNA, Small Interfering; Structure-Activity Relationship; Subcellular Fractions; Substrate Specificity

A rapid increase in the tyrosine phosphorylation of the non-receptor tyrosine kinase FAK is a prominent early event in fibroblasts stimulated by a variety of signaling molecules. However, a variety of epithelial cells, including intestinal epithelial cells, show a high basal level of tyrosine phosphorylated FAK that is only slightly further increased by addition of G protein-coupled receptor (GPCR) agonists or growth factors. In this study, we determined whether these stimuli could elicit FAK phosphorylation at serine residues, including Ser-910 and Ser-843. Our results show that multiple agonists including angiotensin II (ANGII), lysophosphatidic acid (LPA), phorbol esters and EGF induced a striking stimulation of FAK phosphorylation at Ser-910 in rat intestinal epithelial IEC-18 cells via an ERK-dependent pathway. In striking contrast, none of these stimuli promoted a significant further increase in FAK phosphorylation at Tyr-397 in these cells. These results were extended using cultures of polarized human colonic epithelial T84 cells. We found that either carbachol or EGF promoted a striking ERK-dependent phosphorylation of FAK at Ser-910, but these agonists caused only slight stimulation of FAK at Tyr-397 in T84 cells. In addition, we demonstrated that GPCR agonists also induced a dramatic increase of FAK phosphorylation at Ser-843 in either IEC-18 or T84 cells. Our results indicate that Ser-910 and Ser-843, rather than Tyr-397, are prominent sites differentially phosphorylated in response to neurotransmitters, bioactive lipids, tumor promoters and growth factors in intestinal epithelial cells.

Topics: Angiotensins; Animals; Carbachol; Cell Line; Epidermal Growth Factor; Extracellular Signal-Regulated MAP Kinases; Focal Adhesion Protein-Tyrosine Kinases; Humans; Intestinal Mucosa; Ionomycin; Kinetics; Lysophospholipids; Phosphorylation; Protein Kinase C; Rats; Receptors, G-Protein-Coupled; Serine; Signal Transduction; Tyrosine