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Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.

Watson RL, Buck J, Levin LR, Winger RC, Wang J, Arase H, Muller WA - J. Exp. Med. (2015)

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.

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Affiliation: Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60208.

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CD99 cytoplasmic tail mediates TEM through a positively charged juxtamembrane region. (a) Diagram of CD99 cytoplasmic tail and CD99-GFP constructs. (b) Endogenous CD99 in HUVECs was knocked down using CD99 shRNA. CD99-GFP constructs were then reexpressed. Samples treated in parallel were lysed and used for immunoblot analysis of CD99 to assess degree of reexpression and knockdown. (c) Quantitative TEM assays were then performed on knockdown samples. (d) CD99-GFP constructs were overexpressed in iHUVECs. CD99 was then immunoprecipitated from the surface of cells (ensuring only fully processed CD99 was being analyzed). Immunoblot analysis for ezrin, sAC, and CD99 was then performed. (e and f) Quantification of results above. Amount of ezrin (e) and sAC (f) in each sample was normalized to the amount of CD99 immunoprecipitated (to account for any variability in pull-down efficiency between samples). Values were then normalized to wild-type CD99-GFP condition. Images are representative three (b and d) independent experiments. Numerical values are the mean of three (c, e, and f) independent experiments. Error bars represent SD (e and f) or SEM (b; ***, P < 0.001; ****, P < 0.0001; ANOVA).
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fig8: CD99 cytoplasmic tail mediates TEM through a positively charged juxtamembrane region. (a) Diagram of CD99 cytoplasmic tail and CD99-GFP constructs. (b) Endogenous CD99 in HUVECs was knocked down using CD99 shRNA. CD99-GFP constructs were then reexpressed. Samples treated in parallel were lysed and used for immunoblot analysis of CD99 to assess degree of reexpression and knockdown. (c) Quantitative TEM assays were then performed on knockdown samples. (d) CD99-GFP constructs were overexpressed in iHUVECs. CD99 was then immunoprecipitated from the surface of cells (ensuring only fully processed CD99 was being analyzed). Immunoblot analysis for ezrin, sAC, and CD99 was then performed. (e and f) Quantification of results above. Amount of ezrin (e) and sAC (f) in each sample was normalized to the amount of CD99 immunoprecipitated (to account for any variability in pull-down efficiency between samples). Values were then normalized to wild-type CD99-GFP condition. Images are representative three (b and d) independent experiments. Numerical values are the mean of three (c, e, and f) independent experiments. Error bars represent SD (e and f) or SEM (b; ***, P < 0.001; ****, P < 0.0001; ANOVA).

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.


Endothelial CD99 signals through soluble adenylyl cyclase and PKA to regulate leukocyte transendothelial migration.

Watson RL, Buck J, Levin LR, Winger RC, Wang J, Arase H, Muller WA - J. Exp. Med. (2015)

CD99 cytoplasmic tail mediates TEM through a positively charged juxtamembrane region. (a) Diagram of CD99 cytoplasmic tail and CD99-GFP constructs. (b) Endogenous CD99 in HUVECs was knocked down using CD99 shRNA. CD99-GFP constructs were then reexpressed. Samples treated in parallel were lysed and used for immunoblot analysis of CD99 to assess degree of reexpression and knockdown. (c) Quantitative TEM assays were then performed on knockdown samples. (d) CD99-GFP constructs were overexpressed in iHUVECs. CD99 was then immunoprecipitated from the surface of cells (ensuring only fully processed CD99 was being analyzed). Immunoblot analysis for ezrin, sAC, and CD99 was then performed. (e and f) Quantification of results above. Amount of ezrin (e) and sAC (f) in each sample was normalized to the amount of CD99 immunoprecipitated (to account for any variability in pull-down efficiency between samples). Values were then normalized to wild-type CD99-GFP condition. Images are representative three (b and d) independent experiments. Numerical values are the mean of three (c, e, and f) independent experiments. Error bars represent SD (e and f) or SEM (b; ***, P < 0.001; ****, P < 0.0001; ANOVA).
© Copyright Policy - openaccess
Related In: Results  -  Collection

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fig8: CD99 cytoplasmic tail mediates TEM through a positively charged juxtamembrane region. (a) Diagram of CD99 cytoplasmic tail and CD99-GFP constructs. (b) Endogenous CD99 in HUVECs was knocked down using CD99 shRNA. CD99-GFP constructs were then reexpressed. Samples treated in parallel were lysed and used for immunoblot analysis of CD99 to assess degree of reexpression and knockdown. (c) Quantitative TEM assays were then performed on knockdown samples. (d) CD99-GFP constructs were overexpressed in iHUVECs. CD99 was then immunoprecipitated from the surface of cells (ensuring only fully processed CD99 was being analyzed). Immunoblot analysis for ezrin, sAC, and CD99 was then performed. (e and f) Quantification of results above. Amount of ezrin (e) and sAC (f) in each sample was normalized to the amount of CD99 immunoprecipitated (to account for any variability in pull-down efficiency between samples). Values were then normalized to wild-type CD99-GFP condition. Images are representative three (b and d) independent experiments. Numerical values are the mean of three (c, e, and f) independent experiments. Error bars represent SD (e and f) or SEM (b; ***, P < 0.001; ****, P < 0.0001; ANOVA).
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.

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.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL 60208.

Show MeSH
Related in: MedlinePlus