Identification of Fer tyrosine kinase localized on microtubules as a platelet endothelial cell adhesion molecule-1 phosphorylating kinase in vascular endothelial cells.
Platelet endothelial adhesion molecule-1 (PECAM-1) is a part of intercellular junctions and triggers intracellular signaling cascades upon homophilic binding. The intracellular domain of PECAM-1 is tyrosine phosphorylated upon homophilic engagement. However, it remains unclear which tyrosine kinase phosphorylates PECAM-1. We sought to isolate tyrosine kinases responsible for PECAM-1 phosphorylation and identified Fer as a candidate, based on expression cloning. Fer kinase specifically phosphorylated PECAM-1 at the immunoreceptor tyrosine-based inhibitory motif. Notably, Fer induced tyrosine phosphorylation of SHP-2, which is known to bind to the immunoreceptor tyrosine-based inhibitory motif of PECAM-1, and Fer also induced tyrosine phosphorylation of Gab1 (Grb2-associated binder-1). Engagement-dependent PECAM-1 phosphorylation was inhibited by the overexpression of a kinase-inactive mutant of Fer, suggesting that Fer is responsible for the tyrosine phosphorylation upon PECAM-1 engagement. Furthermore, by using green fluorescent protein-tagged Fer and a time-lapse fluorescent microscope, we found that Fer localized at microtubules in polarized and motile vascular endothelial cells. Fer was dynamically associated with growing microtubules in the direction of cell-cell contacts, where p120catenin, which is known to associate with Fer, colocalized with PECAM-1. These results suggest that Fer localized on microtubules may play an important role in phosphorylation of PECAM-1, possibly through its association with p120catenin at nascent cell-cell contacts.
Kogata
Naoko
N
Department of Structural Analysis, National Cardiovascular Center Research Institute, Suita, Osaka 565-8565, Japan.
Masuda
Michitaka
M
Kamioka
Yuji
Y
Yamagishi
Akiko
A
Endo
Akira
A
Okada
Masato
M
Mochizuki
Naoki
N
D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D051177:Catenins; D002417:Cattle; D002448:Cell Adhesion / Q000502:physiology; D015815:Cell Adhesion Molecules / Q000378:metabolism / Q000502:physiology; D002478:Cells, Cultured; D003001:Cloning, Molecular; D042783:Endothelial Cells / Q000201:enzymology* / Q000502:physiology; D015723:Gene Library; D049452:Green Fluorescent Proteins; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D008870:Microtubules / Q000201:enzymology* / Q000378:metabolism / Q000502:physiology; D009154:Mutation; D010750:Phosphoproteins / Q000378:metabolism* / Q000502:physiology; D010766:Phosphorylation; D019408:Platelet Endothelial Cell Adhesion Molecule-1 / Q000378:metabolism* / Q000502:physiology; D017434:Protein Structure, Tertiary; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism / Q000502:physiology; D011505:Protein-Tyrosine Kinases; D011518:Proto-Oncogene Proteins / Q000378:metabolism* / Q000502:physiology; D000095002:Delta Catenin
D048868:Adaptor Proteins, Signal Transducing; D051177:Catenins; D015815:Cell Adhesion Molecules; C098587:GAB1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008164:Luminescent Proteins; D010750:Phosphoproteins; D019408:Platelet Endothelial Cell Adhesion Molecule-1; D011518:Proto-Oncogene Proteins; C052602:proto-oncogene protein c-fes-fps; D049452:Green Fluorescent Proteins; D011505:Protein-Tyrosine Kinases; C516724:PTPN11 protein, human; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; D000095002:Delta Catenin