In view of a substantial amount of data showing that tyrosine phosphorylation destabilizes adherens junctions (see Introduction) it was surprising to find that pervanadate treatment stabilized desmosomes, increasing their adhesiveness and resistance to calcium chelation. This was especially so since previous studies involving activation/inhibition of EGF signalling, though not producing dramatic effects, indicated that tyrosine phosphorylation tended to destabilize desmosomal adhesion.17–19
On the other hand, the work of Miravet et al. (2003) showed that phosphorylation of Pg at different sites can stabilize or destabilize its interaction with adherens junction components.20
It is also of interest that the non-receptor tyrosine kinase Fyn, which phosphorylates β-catenin and Pg, has been associated with the promotion of keratinocyte adhesion.25
Taken together with previous observations our results suggest that tyrosine phosphorylation can have opposing effects on desmosomal adhesion.
Our biochemical analysis shows that pervanadate induced tyrosine phosphorylation of Dsg2 and Pg in both the NP40-soluble and -insoluble fractions. This contrasts with the work of Gaudry et al.17
who found Pg tyrosine phosphorylated through the EGF receptor exclusively in the non-ionic detergent-soluble fraction. The proteins in the insoluble fraction are presumably largely or entirely located in desmosomes, a suggestion consistent with our observation that exogenous HA-tagged Dsg2 was co-localized with the desmosomal plaque marker DP by confocal microscopy. Indeed, Kimura et al.2
showed that the molecular composition of both calcium dependent and hyper-adhesive desmosomes to be identical. In particular, both contained Pg and desmoplakin. This again appears to contrast with the work of Gaudry et al.17
who found no association between Pg tyrosine phosphorylated through the EGF receptor and desmoplakin. A further contrast between our results and those obtained with the EGF signalling system is that inhibition of the EGF receptor caused initiation of desmosome assembly even in low calcium medium,19
whereas pervanadate-induced phosphorylation rendered desmosomes resistant to calcium chelation.
Tyrosine phosphorylation of desmosomes has previously been demonstrated by immunogold labelling and electron microscopy.26
This is important because it is clearly phosphorylation events that occur in desmosomes that directly regulate their adhesiveness. We have shown previously that PKCα, activation of which converts desmosomes from the hyper-adhesive form to calcium dependence, is a component of the cytoplasmic plaques of calcium-dependent desmosomes.5
We may speculate that a tyrosine kinase and phosphatase are also located in the desmosomal plaque under some circumstances. Furthermore, it appears that both serine/threonine and tyrosine phosphorylation can be involved in regulating desmosomal adhesion, but the former destabilizes and latter can stabilize.
Desmosomal components present in the NP40-soluble fraction represent materials that are en route for either incorporation into desmosomes or degradation. Our finding of small amounts of tyrosine-phosphorylated Dsg2 and Pg in the soluble fraction of control cells suggests that tyrosine phosphorylation may be normally involved in one or other of these processes. Results from pervanadate-treated cells did not give a clear indication of whether an involvement in degradation or desmosomes assembly is more likely. We consistently found slightly less Dsg2 in the soluble fraction of treated cells than in controls, whereas the amount in the insoluble fraction remained constant. This might suggest an involvement in Dsg2 degradation since there was no evidence for increased incorporation into the insoluble fraction. On the other hand, the amount of Pg that co-precipitated with Dsg2 remained constant between control and treated cells, suggesting that tyrosine phosphorylation did not cause disruption of the Dsg2-Pg complex, but if anything enhanced it.
We conclude that the involvement of tyrosine phosphorylation in regulation of desmosomal adhesion is complex. On the one hand activation of EGF signalling destabilizes desmosomes; on the other, pervanadate treatment stabilizes them. Further work will be required in order to elucidate the subtleties of the mechanisms involved in these opposite responses to tyrosine phosphorylation of desmosomal components. Because of the involvement of desmosomes in human disease, including cancer27–29
and in morphogenesis30,31
such investigations are strongly merited.