Previous studies have shown that CDCP1 might regulate cell–matrix adhesion and be involved in metastasis. The results presented here show that CDCP1 phosphorylation is correlated with the phosphorylation of proteins known to regulate adhesion and motility. Furthermore, using antibody stimulation to explore signaling events initiated by CDCP1 phosphorylation, we confirmed its involvement in the regulation of cell adhesion and motility. Most striking is our observation that both in human tissue and upon acute activation in cell culture, the tyrosine phosphorylation of CDCP1 is correlated with SFK activation and with inhibition of FAK.
It is a paradox that CDCP1 activates SFKs, resulting in increased phosphorylation of known SFK substrates such as SHC and SHP2, yet causes a decrease in FAK phosphorylation. Of note, whereas SHP2 is known to regulate FAK tyrosine phosphorylation in other contexts, suppression of SHP2 expression by RNA interference did not diminish the effect of CDCP1 on FAK phosphorylation (data not shown). A possible explanation is that CDCP1 recruits SFKs away from integrins and FAK, and activates these enzymes at a new location in areas of cell–cell contact where distinct substrates are engaged. This model is consistent with the observation that CDCP1 engagement modulates cortical actin at regions of cell–cell contact and decreases interactions with FN. Although siRNA against CDCP1 decreases the phosphorylation of SFKs, it does not completely eliminate SFK phosphorylation, so it is likely that the increased FAK phosphorylation under these conditions is because of a localized pool of active SFKs. Recruitment of SRC to FAK has been shown to be necessary to initiate FAK tyrosine phosphorylation, although SRC activity appears dispensable in at least some instances (Brunton et al., 2005
The inhibition of adhesion seen here upon acute activation of CDCP1 is consistent with observations that cells overexpressing CDCP1 show a rounded morphology (Bhatt et al., 2005
), and with a correlation between CDCP1 tyrosine phosphorylation and loss of adhesion observed in some contexts (Spassov et al., 2009
). Moreover, Liu et al. (2011)
have shown recently that CDCP1 overexpression reduces cell–matrix adhesion. However, in contrast to some of these other reports, in our experience, detaching cells from their substratum (using a non-enzymatic method that does not induce CDCP1 cleavage) does not lead to CDCP1 phosphorylation within 2 h (see ). However, we did observe that a high concentration of EGTA (ethylene glycol-bis(β-aminoethyl ether)-N
,-tetraacetic acid) (>2mM) induced a rapid and transient activation of CDCP1 tyrosine phosphorylation (unpublished observation). In addition, Alvares et al. (2008)
reported recently that mAb-induced phosphorylation of CDCP1 could not be induced in suspended cells; however, adhesion status appears to make little or no difference under our conditions (). Although some of these discrepancies are likely because of the use of different cell lines, it would be interesting to understand better the possible crosstalk between CDCP1 activation and cell–matrix adhesion. In a recent study, overexpression of CDCP1 was shown to promote loss of cell–matrix adhesion and FAK phosphorylation, events correlated with impairment in integrin clustering. These results and others in that study addressing the correlation of FAK and CDCP1 phosphorylation are quite consistent with ours (Spassov et al., 2011
), and suggest that acute activation of CDCP1 might induce changes in cell–matrix adhesion by affecting integrin clustering. This supports the concept that actin polymerization changes or recruitment of actin filament might underlie a number of functional outcomes of CDCP1 activation. In keeping with this, another recent report has identified cortactin as an interacting partner of PKCδ, and suggests that the PKCδ–cortactin complex mediates changes in migration downstream from CDCP1 phosphorylation (Miyazawa et al., 2010
). Although we do observe tyrosine-phosphorylated PKCδ (Supplementary Figure 7S
) at cell–cell contacts following CDCP1 activation, our preliminary results have shown that suppression of PKCδ expression only modestly affects actin recruitment at cell–cell contacts in response to CDCP1 activation.
While we observe increased cell motility in Transwell assays upon CDCP1 activation, we also found that CDCP1 activation correlated with the recruitment of actin at the periphery of the cell and faster junction formations in calcium switch experiments. These two sets of events seem somewhat contradictory. As cortical actin accumulation occurs upon maturation of cell–cell contacts, this suggests that CDCP1 activation could reinforce cell–cell adhesion. Interestingly, in the Transwell assay, cells migrate after seeding without prior establishment of a monolayer. Thus, the effect of CUB1 on adhesion to the substratum might overcome the effect on cell–cell contacts in this assay. In fact, activation of CDCP1 had no effect on the healing of the scratched cell monolayer in multiple cell lines (data not shown). This is in contrast to a recent report showing that CDCP1 knockdown in MCF10A cells prevents cell migration in the same setting (Spassov et al., 2011
). Moreover, as expected from the fact that CDCP1 is not tyrosine-phosphorylated in unstimulated MCF10A cells or after wounding of an epithelial monolayer, knockdown of CDCP1 does not have any effect on cell migration or cell–cell contacts in our experience (CHB, unpublished studies). It remains unclear at this point why our results and those of Spassov et al. (2011)
are disparate in that regard, as their study also shows that CDCP1 is not tyrosine-phosphorylated in attached, unstimulated cells.
It is possible that CDCP1 is involved in the coordination of cell–matrix and cell–cell adhesion in the normal epithelia. On the other hand, downregulation of E-cadherin and loosening of cell–cell adhesion, as seen frequently in carcinomas or during epithelial-to-mesenchymal transition, could result in a context where CDCP1 activation produces mainly an increase in cell motility. The endogenous mechanism(s) of CDCP1 activation remain unknown, although proteolysis by matripase or other serine proteases represents an attractive mechanism (He et al., 2010
) especially in the context of wound-healing. In this context, our results suggest that CDCP1 might be involved in coordinating cellular adhesion to the extracellular matrix and to other cells.
Our findings concerning downregulation of CDCP1 by prolonged exposure to anti-CDCP1 antibodies have direct relevance to reports that mAbs against CDCP1 block experimental metastasis (Uekita et al., 2008
; Deryugina et al., 2009
). Our observations bolster the idea that anti-CDCP1 antibodies could be used to promote the down-regulation of CDCP1 and CDCP1/SFK complexes in some cancers, as these reports suggested, and indicate a mechanism (CDCP1 downregulation) by which this occurs.
A comparison of the results obtained in cell culture assays with the phospho-tyrosine profile from human tissue strongly suggests that the signaling events defined using specific CDCP1 activation in cell culture are indeed operating in human tissue. This supports the notion that biomarkers for SFK activity should be chosen carefully depending on the CDCP1 status. In particular, SFK autophosphorylation, together with the phosphorylation status of FAK and of SRC/FAK substrates, has been proposed as a reporter for SFK activity in tumors, based on the fact that SFK activity is best correlated with changes in migration in cell culture models of carcinomas (Brunton et al., 2005
; McLean et al., 2005
; Serrels et al., 2006
). However, in cases where CDCP1 is involved in the regulation of SFK, high levels of SFK activity might not always correlate well with high levels of FAK and SFK/FAK substrate phosphorylation.