Cell Shrinkage Regulates Src Kinases and Induces Tyrosine Phosphorylation of Cortactin, Independent of the Osmotic Regulation of Na+/H+ Exchangers

Abstract

The signaling pathways by which cell volume regulates ion transporters, e.g. Na ؉ /H؉ exchangers (NHEs), and affects cytoskeletal organization are poorly understood. We have previously shown that shrinkage induces tyrosine phosphorylation in CHO cells, predominantly in an 85-kDa band. To identify volume-sensitive kinases and their substrates, we investigated the effect of hypertonicity on members of the Src kinase family. Hyperosmolarity stimulated Fyn and inhibited Src. Fyn activation was also observed in nystatin-permeabilized cells, where shrinkage cannot induce intracellular alkalinization. In contrast, osmotic inhibition of Src was prevented by permeabilization or by inhibiting NHE-1. PP1, a selective Src family inhibitor, strongly reduced the hypertonicity-induced tyrosine phosphorylation. We identified one of the major targets of the osmotic stresselicited phosphorylation as cortactin, an 85-kDa actinbinding protein and well known Src family substrate. Cortactin phosphorylation was triggered by shrinkage and not by changes in osmolarity or pH i and was abrogated by PP1. Hyperosmotic cortactin phosphorylation was reduced in Fyn-deficient fibroblasts but remained intact in Src-deficient fibroblasts. To address the potential role of the Src family in the osmotic regulation of NHEs, we used PP1. The drug affected neither the hyperosmotic stimulation of NHE-1 nor the inhibition of NHE-3. Thus, members of the Src family are volumesensitive enzymes that may participate in the shrinkage-related reorganization of the cytoskeleton but are probably not responsible for the osmotic regulation of NHE.

Transforming Growth Factor β2 inhibition of Hepatocyte Growth Factor-induced endothelial proliferation and migration

“…To understand how TGF b 2 inhibits HGF, we elucidated which intracellular signalling pathways are able to discriminate between HGF-induced cell migration and proliferation in endothelial cells. In this regard, it is noteworthy that PP1, a selective Src family inhibitor (Kapus et al, 1999), strongly reduced HUVEC migration in response to HGF, while it did not signi®cantly inhibit cell proliferation. Interestingly, we found that TGF b 2 blocks HGF-dependent tyrosine phosphorylation of Src.…”

“…We observed that HGF did not induce tyrosine phosphorylation of Src in the presence of TGF b 2 (Figure 5a). Accordingly, PP1, which is a selective pyrazolo pyrimidine-type inhibitor of Src (Kapus et al, 1999), inhibited HGF-induced cell migration at a concentration as low as 5 mM (Figure 5b), while it did not signi®cantly alter cell proliferation (Figure 5c). …”

Cortactin: coupling membrane dynamics to cortical actin assembly

“…Hyperosmolarity elevates tyrosine phosphorylation of cortactin as well as the activity of the Fyn, while suppressing Src activity (Kapus et al, 1999). The requirement for Fyn in this process is further underscored by the reduction of cortactin tyrosine phosphorylation in hyperosmotic Fyn 7/7 , but not Src 7/7 cells (Kapus et al, 1999). A rapid reduction in cell volume also activates Fer, which is suppressed by pretreatment with the Src family inhibitor PP1 as well as in Fyn 7/7 cells (Kapus et al, 2000).…”

“…Other evidence supporting a role for Fer in cortactin tyrosine phosphorylation has come from the analysis of signaling events involved in cell shrinkage. Hyperosmolarity elevates tyrosine phosphorylation of cortactin as well as the activity of the Fyn, while suppressing Src activity (Kapus et al, 1999). The requirement for Fyn in this process is further underscored by the reduction of cortactin tyrosine phosphorylation in hyperosmotic Fyn 7/7 , but not Src 7/7 cells (Kapus et al, 1999).…”

Making and breaking contacts: the cellular biology of cadherin regulation

“…It should be noted, however, that many non-specific manuevers that inhibit endocytosis can influence cadherin function by alternative mechanisms. For example, hypertonic stress induces tyrosine phosphorylation of cortactin [22], which contributes to cadherin junction integrity [23,24] (Helwani et al, unpublished). These disparate models thus remain to be resolved and warrant further investigation.…”