Signal transduction via transmembrane receptor tyrosine kinases often involves the activation of additional downstream kinases, both serine/threonine-directed and tyrosine-directed. A number of studies have implicated Src-family kinases in tyrosine phosphorylation of caveolin-1. Caveolin-1 was originally identified as a major tyrosine-phosphorylated protein in v-Src-transformed cells [1,39,59,60]. Caveolin is also highly phosphorylated in cells that overexpress c-Src or c-Fyn, and is one of the most prominent phosphoproteins detected in these cells [15,16,31,61]. Caveolin-1 binds to and co-purifies with Src-family kinases [10,52,62,63]. Furthermore, caveolin-1 can be directly phosphorylated by Src and Fyn in vitro [16,64]. These data indicated that Src and Fyn are caveolin tyrosine kinases in cells.
Caveolin-1 is phosphorylated in response to a number of cellular stresses, including shear stress, oxidative stress, and osmotic shock [24,28-32,34]. These stresses also activate Src-family kinases, including both Src and Fyn [31,34,65-67]. While overexpression of either kinase is sufficient to cause caveolin tyrosine phosphorylation under basal conditions, overexpression of Fyn also caused significant hyperphosphorylation of caveolin-1 in response to stress . Therefore, whilst overexpression can activate Fyn to some extent, stress clearly activates the kinase. These data indicate that Fyn is part of the signaling cascade leading to caveolin tyrosine phosphorylation in response to stress. Consistent with this, expression of kinase-inactive Src blocked caveolin phosphorylation in response to osmotic shock . Furthermore, the small molecule Src-family kinase inhibitors SU6656, PP2, and PD180970 inhibited both oxidative stress and osmotic shock-induced caveolin phosphorylation, indicating that both require activation of a Src-family kinase . Shear stress-induced caveolin phosphorylation was blocked by the small molecule Src-family kinase inhibitor PP1, and enhanced by inactivation of Csk, a negative regulator of Src-family kinases . Consistent with the inhibitor data, both oxidative stress and osmotic shock-induced caveolin phosphorylation were blocked in SYF-/- cells, a cell line derived from a knockout animal deficient in three Src-family kinases: Src, Yes, and Fyn . These data show that stress-induced caveolin phosphorylation requires activation of a Src-family kinase.
Using cell lines derived from single kinase knockout mice (Src-/-, Yes-/-, and Fyn-/-), it was found that expression of Fyn is required for both oxidative stress and osmotic shock-induced caveolin phosphorylation, while expression of Src and Yes are not . In fact, phosphorylation in the Src-/- cells was even higher than that observed in the cells from wild-type mice. This correlated with an increase in Fyn expression in these cells relative to wild-type cells. Heterologous expression of Fyn was sufficient to restore oxidative stress-induced caveolin phosphorylation in both the Fyn-/- and SYF-/- cells, indicating that Fyn can promote the phosphorylation of caveolin in the absence of Src or Yes. Therefore, Fyn expression is both necessary and sufficient for stress-induced caveolin phosphorylation.
Both Src and Fyn can directly phosphorylate caveolin, and both are activated in response to stress, but only Fyn is required for stress-induced caveolin phosphorylation. These data suggest that the Src that is activated in response to stress is localized to a compartment that does not contain caveolin . Fyn is targeted to caveolae/lipid rafts due to tandem acylation by myristate and palmitate. Co-localization of dually acylated proteins such as Lyn or Fyn with caveolin has recently been confirmed using fluorescence resonance energy transfer (FRET) . In contrast to Fyn, Src is only singly acylated with myristate. Singly acylated proteins have less affinity for membranes and do not co-purify with caveolae/lipid rafts [52,68]. The behavior of singly acylated proteins, such as Src, was not determined in the FRET experiment. However, Src can bind directly to caveolin . The present data suggest that there are distinct signaling complexes containing caveolin and Fyn or caveolin and Src that are differentially activated in response to stress. In the context of cellular stress, Fyn - but not Src - is a caveolin tyrosine kinase. However, Src may phosphorylate caveolin under other types of stimuli, for example during cell attachment and spreading on fibronectin .
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