PECAM-1 functions as an adhesive molecule and as a signaling mediator during cell-cell contact (Newman, 1997
). Phosphorylation of PECAM-1 is required for triggering intracellular signaling (Newman, 1999
). Herein, we have identified Fer tyrosine kinase as a PECAM-1 phosphorylating kinase and as an inducer for SHP-2 and Gab1 phosphorylation. In addition, we have suggested an important role in localization of Fer to microtubules for phosphorylation of PECAM-1.
Bacterial expression cloning enabled us to isolate Fer kinase as a PECAM-1 phosphorylating kinase. This method is advantageous in screening kinases that directly phosphorylate the cytoplasmic domain of PECAM-1. Previous data have shown that Src is capable of phosphorylating and binding to PECAM-1 by in vitro kinase assays and in an overexpression study (Lu et al., 1997
; Cao et al., 1998
). In our laboratory, phosphorylation of PECAM-1 induced by mechanical stretch was not perturbed in the presence of the Src family kinase inhibitor PP2 (unpublished observation in Osawa et al., 2002
). Thus, Fer seems to be the best candidate for a PECAM-1–phosphorylating kinase. This notion is supported by the evidence that Tyr663 of PECAM-1 is a preferred site for Fer (), whereas Tyr686 is a preferred site for Src and Csk families of protein tyrosine kinase (Cao et al., 1998
). Because mice harboring inactivating mutation of Fer are viable (Craig et al., 2001
), Fes and/or LynB, both of which have been shown to phosphorylate PECAM-1, may compensate for the defects in these mice.
The physiological consequence of PECAM-1 phosphorylation remains unknown. We previously demonstrated that phosphorylated PECAM-1 triggered SHP-2-mediated intracellular signaling, including recruitment of Gab1 and ERK activation in vascular endothelial cells (Masuda et al., 1997
; Osawa et al., 2002
). In the present study, we have shown that Fer can induce phosphorylation of both SHP-2 and Gab1 (). Moreover, Gab1 and SHP-2 cooperatively function to activate ERK and to induce branching morphogenesis, thereby triggering active cell motility in Madin-Darby canine kidney cells (Schaeper et al., 2000
). ERK is involved in actomyosin contractility, which is essential for cell migration, by phosphorylating myosin light chains (Cheresh et al., 1999
). In addition, cortactin, a substrate of Fer kinase, participates in membrane ruffling by binding to F-actin (Kim and Wong, 1998
). A deficiency of cortactin phosphorylation and a migration defect were found in Fer-deficient fibroblasts and in Fer-deficient mast cells, respectively (Craig et al., 2001
; Craig and Greer, 2002
). Collectively, Fer seems to orchestrate the downstream signaling of PECAM-1, leading to cell migration by phosphorylating signaling molecules regulating cell motility.
Most cytoplasmic protein tyrosine kinases contain conserved domains such as SH2, SH3, and pleckstrin homology besides tyrosine kinase domain (Blume-Jensen and Hunter, 2001
). Among protein tyrosine kinases, Fer and Fes are the only family members that contain an FCH domain, the function of which has not yet been defined. Because the FCH domain of CIP4 binds to microtubules (Tian et al., 2000
), the amino-terminal FCH of Fer and Fes has been expected to function as a microtubule-targeting domain (Greer, 2002
). We found that GFP-tagged Fer and Fes partially colocalized with microtubules and demonstrated that microtubule targeting of Fer was perturbed by deletion of the coiled-coil domain. Intriguingly, the removal of the FCH domain from Fer did not perturb the localization of Fer to microtubules, as shown in . These data indicate that the FCH domain is not the sole microtubule-targeting domain. EGFP-tagged Fer localized at both proximal and peripheral microtubules in the polarizing zone toward the leading edge. In contrast, Fes localized at proximal microtubules. The distinct localization of Fer and Fes may account for efficiency of the PECAM-1 phosphorylation. Moreover, the evidence that Fer localized on vesicular structures near proximal microtubules such as GFP-tagged Fes () coincides with the previous report demonstrating the localization of Fes/Fps to vesicular structures and partial colocalization with several Rab proteins (Zirngibl et al., 2001
We have demonstrated for the first time that Fer localizes to microtubules, as shown in . We developed an antibody against Fer to examine the localization of endogenous Fer in endothelial cells. Endogenous Fer could not be detected on microtubules by using the antibody we developed, raising two possibilities; one is that endogenous Fer that is inactive without any stimulation may not localize to microtubules, and the other is simply that the antibody is not sensitive enough to detect the endogenous Fer. The former possibility is supported by the evidence that overexpressed Fer is autophosphorylated in fibroblasts (Rosato et al., 1998
) and our data that EGFP-tagged WT Fer but not KD Fer localized to microtubules. In contrast, Hao et al.
, 1991 and Yates et al.
, 1995 reported that Fer and Fes localize in the nucleus. Although our results did not coincide with these previous reports, the localization of Fer may depend on whether PECAM-1 is activated.
Microtubules are polarized in the migrating cells and grow forward to the leading edge (Wittmann and Waterman-Storer, 2001
). In vascular endothelial cells, microtubules detected by EGFP-tagged Fer grew toward neighboring cells and reached cell-cell contacts, where PECAM-1 accumulated (Figures and ). As shown in , p120ctn also accumulated at nascent cell-cell contacts toward which microtubules were growing. p120ctn can associate with Fer via its coiled-coil domain (Kim and Wong, 1995
). In addition, we found that dCC2 lacking p120ctn association-domain phosphorylated PECAM-1 to a lesser extent than WT (). Thus, an association between Fer and p120ctn at nascent cell-cell contacts seems to contribute to phosphorylation of PECAM-1.
In conclusion, we identified Fer tyrosine kinase as a PECAM-1–phosphorylating kinase and demonstrated for the first time that Fer localizes to microtubules in the polarizing vascular endothelial cells. The microtubule-targeted Fer seems to be essential for phosphorylation of PECAM-1 by p120ctn-mediated recruitment to cell-cell contacts.