As human lung microvascular endothelia achieve confluence, src family kinases are activated, and tyrosine-phosphorylated p120 catenin physically couples NEU1 sialidase to CD31.
In postconfluent human pulmonary microvascular endothelial cell (HPMEC)s, NEU1 sialidase associates with and desialylates the src family kinase (SFK) substrate, CD31, and disrupts angiogenesis. We asked whether the NEU1-CD31 interaction might be SFK-driven. We found that normalized phospho-SFK (PY416) signal is increased in postconfluent HPMECs compared to subconfluent cells and prior SFK inhibition with PP2 or SU6656 completely blocked NEU1 association with and desialylation of CD31. Prior silencing of each of the four SFKs expressed in HPMECs, as well as CD31, dramatically reduced confluence-induced SFK activation. No increases in tyrosine phosphorylation of NEU1 or CD31 were detected. However, in postconfluent cells, we found increased tyrosine phosphorylation of a 120 kDa protein that was identified as p120 catenin (p120ctn). Prior silencing of c-src, fyn, or yes each reduced p120ctn phosphorylation. Prior knockdown of p120ctn prevented NEU1-CD31 association in both co-immunoprecipitation and pull-down assays. In these same assays, p120ctn associated with each of the four HPMEC-expressed SFKs as well as CD31 and NEU1. The CD31-p120ctn interaction was SFK-dependent whereas the NEU1-p120ctn interaction was not. Using purified recombinant binding partners in a cell-free system, direct protein-protein interactions between NEU1, CD31, and p120ctn were detected. Our combined data indicate that as HPMECs achieve confluence and CD31 ectodomains become homophilically engaged, multiple SFKs are activated to increase tyrosine phosphorylation of p120ctn, which in turn, functions as a cross-bridging adaptor molecule that physically couples NEU1 to CD31, permitting NEU1-mediated desialylation of CD31. These findings establish a SFK-driven, p120ctn-dependent mechanism for NEU1 recruitment to CD31.
U.S. Department of Veterans Affairs, Baltimore, MD 21201, United States; Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
Sang W
SW
Hyun
U.S. Department of Veterans Affairs, Baltimore, MD 21201, United States; Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
Anguo
A
Liu
U.S. Department of Veterans Affairs, Baltimore, MD 21201, United States; Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
Zhenguo
Z
Liu
Department of Pediatrics, University of Maryland School of Medicine, Baltimore, MD 21201, United States.
Erik P
EP
Lillehoj
Department of Pathology, Yale University School of Medicine, New Haven, CT 06520, United States.
Joseph A
JA
Madri
Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232, United States.
Albert B
AB
Reynolds
U.S. Department of Veterans Affairs, Baltimore, MD 21201, United States; Department of Medicine, University of Maryland School of Medicine, Baltimore, MD 21201, United States. Electronic address: sgoldblu@mbrc.umaryland.edu.
Simeon E
SE
Goldblum
D051177:Catenins / Q000235:genetics* / Q000378:metabolism; D002460:Cell Line; D002474:Cell-Free System; D042783:Endothelial Cells / Q000378:metabolism; D006801:Humans; D008168:Lung / Q000378:metabolism; D055806:Microvessels / Q000378:metabolism; D019158:N-Acetylneuraminic Acid / Q000235:genetics / Q000378:metabolism; D018919:Neovascularization, Physiologic / Q000235:genetics; D009439:Neuraminidase / Q000235:genetics* / Q000378:metabolism; D010766:Phosphorylation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000235:genetics* / Q000378:metabolism; D011485:Protein Binding; D060066:Protein Interaction Maps / Q000235:genetics; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics; D051575:Proto-Oncogene Proteins c-yes / Q000235:genetics; D015398:Signal Transduction / Q000235:genetics; D019061:src-Family Kinases / Q000235:genetics; D000095002:Delta Catenin
D051177:Catenins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; C489834:YES1 protein, human; D019061:src-Family Kinases; C486385:NEU1 protein, human; D009439:Neuraminidase; D019158:N-Acetylneuraminic Acid; D000095002:Delta Catenin; C000722347:CTNND1 protein, human
Endothelial cell; Lung; Neuraminidase; Platelet endothelial cell adhesion molecule (PECAM-1); Sialic acid