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: The cytoplasmic tail of CD99 is composed of 39 aa, contains no known signaling motifs, and has no known binding partners. However, within the juxtamembrane cytoplasmic tail there lies a short lysine-rich region that is highly conserved across species (Banting et al., 1989; Ellis et al., 1994; Park et al., 2005). To test the importance of this region for CD99 function, we generated a series of CD99-GFP mutants (Fig. 8 a). Endogenous CD99 was knocked down in HUVECs using shRNA, which decreased TEM significantly. Exogenous CD99-GFP was then reexpressed in these cells. Immunoblot analysis demonstrated sufficient knockdown of endogenous CD99 and comparable levels of reexpression of the rescue constructs (Fig. 8 b). Whereas wild-type CD99-GFP was able to rescue TEM, CD99 lacking the majority of its cytoplasmic tail was unable to do so (Fig. 8 c). Furthermore, mutation of the four juxtamembrane lysine residues (KKKLCFK) to negatively charged glutamic acids (EEELCFE) failed to restore TEM. However, conserving the positive charge of this region by mutating the lysine’s residues to arginines (RRRLCFR) brought TEM to wild-type levels. Two point mutations were also tested: a putative PKC site (SHR) and a casein kinase II phosphorylation site (TLLE). CD99 bearing either of these two mutations fully rescued TEM, demonstrating that these residues were not important for CD99 function in TEM. These data prove that the positive charge of the juxtamembrane lysines in the cytoplasmic tail of CD99 are required for its function during TEM.
Affiliation: Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60208.