Ectopic αIIbβ3 Integrin Signaling Involves 12-Lipoxygenaseand PKC-mediated Serine Phosphorylation Events in Melanoma Cells

Summary

Megakaryocytic genes such as αIIbβ3 can be expressed by malignant cells as part of the disturbances in their gene regulation. However, the function of the gene product greatly depends on the interaction of the ectopic protein with the new environment. The outside-in signaling of the ectopically expressed αIIbβ3 integrin was studied in B16a murine melanoma cells using a monoclonal antibody, specifically directed to the activated conformation of αIIbβ3, PAC-1 and the physiological ligand, fibrinogen. Ligation of αIIbβ3 induced downregulation of FAK but serine phosphorylation of three protein bands, 20/21, 85 and 140 kDa within 1-15 min. Flow cytometry indicated that the ligation of the receptor in B16a cells induces ~50% increase in phosphoserine positive cells within 5-15 min. 12-lipoxygenase is placed downstream in the signaling pathway, since ligation of αIIbβ3 induces 12-HETE production within 5 min and pretreatment of tumor cells with select lipoxygenase inhibitior, Baicalein, prevents the increase in serine phosphorylation. Confocal microscopy of adherent tumor cells demonstrated rearrangement of actin filaments upon αIIbβ3 ligation paralleled by downregulation of p125FAK and phoshotyrosine⁺ adhesion plaques and translocation of PKCα to stress fibers and cortical actin. PKC appears to be the major effector serine kinase of the αIIbβ3-coupled signaling pathway, since pretreatment of tumor cells with a select PKC inhibitor, Calphostin C, prevents the ligationinduced serine phosphorylation. Previous studies have indicated a role for the 12-lipoxygenase-PKC signaling pathway in platelet aggregation as well as tumor invasion, therefore the involvement of this cascade in the signaling of the ectopic αIIbβ3 integrin may partially explain its role in tumor progression.

Key words

Melanoma - integrin αIIbβ3 - signal transduction - flow cytometry - confocal microscopy

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