Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.
Bottom Line: How CD99 signals during this process remains unknown.We show that during TEM, endothelial cell (EC) CD99 activates protein kinase A (PKA) via a signaling complex formed with the lysine-rich juxtamembrane cytoplasmic tail of CD99, the A-kinase anchoring protein ezrin, and soluble adenylyl cyclase (sAC).PKA then stimulates membrane trafficking from the lateral border recycling compartment to sites of TEM, facilitating the passage of leukocytes across the endothelium.
Affiliation: Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60208.Show MeSH
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Mentions: We next investigated how CD99 engagement led to increased cAMP and activated PKA. In mammalian cells, two classes of AC catalyze the formation of cAMP: G protein–regulated transmembrane ACs (tmACs) and sACs (sACs). KH7 is an sAC-specific inhibitor, whereas 2’5′-didexoyadenosine (ddAdo), when used at or below 50 µM, is a tmAC-selective inhibitor (Bitterman et al., 2013). HUVECs were pretreated with KH7, ddAdo, or dimethylsulfoxide (DMSO; control) and cross-linked with anti-CD99 or mIgG1 control. Immunoblot analysis for phospho-VASP was performed to assess PKA activity. The activation of PKA by cross-linking CD99 was significantly attenuated in EC treated with KH7, whereas inhibition of tmACs with ddAdo had no effect (Fig. 4, a and b). To confirm this observation, we used antibody-coated beads and IF staining to spatially assess PKA activity. As seen previously, anti-CD99–coated beads were locally enriched with phospho-PKA staining. Furthermore, activation of PKA was significantly blocked in HUVECs pretreated with KH7 but not with ddAdo. As an additional control, we demonstrated that CD99-coated bead activation of PKA was blocked by the PKA inhibitor myristolyated protein kinase inhibitor peptide (PKI; Fig. 4, c–e). From these data, we conclude that CD99 cross-linking activated PKA via stimulation of sAC.
Affiliation: Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60208.