Model of the Interaction of Fyn and Abl in Caveolin Phosphorylation

Expression of both Abl and Fyn is necessary for caveolin phosphorylation, and overexpression of either is sufficient to induce caveolin phosphorylation. Therefore, the caveolin phosphorylation pathway represents a newly identified signaling cascade involving both Abl and Fyn. Signaling complexes involving both Src-family kinases and Abl have also been identified in a number of other systems [82-89]. Whilst both Abl and Fyn can phosphorylate caveolin directly, it is believed that Fyn and Abl act synergistically to induce sustained, high-level phosphorylation of cav-eolin-1. Our model is that Fyn activity is required for the efficient recruitment of Abl to the caveolae, and that Abl is required for sustained phosphorylation of caveolin (Fig. 6.2). In this model, activation of Fyn that is resident in the caveolae leads to phosphorylation of caveolin-1 at Tyr14. This phosphorylation creates a binding site for the SH2 protein binding domain of Abl (consensus binding site, pY-X-X-P), and recruitment of Abl into the complexes (domain structure of Abl: SH3, SH2, kinase domain, tail). Abl then phosphorylates adjacent caveolin molecules in the complex. In this way, the phosphorylation when both kinases are activated is significantly greater than either alone. Overexpression of Abl causes activation of Abl and inefficient phosphorylation of caveolin-1, which leads to the recruitment of Abl into caveolae and more efficient phosphorylation. Overexpression of Fyn bypasses an inherent limitation on the level of caveolin phosphoryla-tion that can normally be catalyzed by Fyn alone (see below). This model may also explain why some stimuli lead to only transient phosphorylation of caveolin, while others lead to sustained phosphorylation of caveolin. Whilst all activate Src-family kinases in caveolae and initiate caveolin phosphorylation, they may differ in their ability to activate and translocate Abl to the caveolae. Our investigations into the signaling cascades that lie downstream of caveolin phosphorylation strongly sup-

Fig. 6.2 Fyn and Abl are caveolin tyrosine kinases. Insulin and stress activate the Src-family kinase Fyn and autophosphorylation of the kinase (pY416). Fyn is localized to lipid rafts via tandem acylation and to caveolae through direct binding to caveolin. Upon activation, Fyn phosphorylates caveolin-1 on Tyr14. Tyrosine phosphorylation promotes SH2 domain-mediated binding of Abl. This in turn leads to the phosphorylation of adjacent caveolin molecules.

Fig. 6.2 Fyn and Abl are caveolin tyrosine kinases. Insulin and stress activate the Src-family kinase Fyn and autophosphorylation of the kinase (pY416). Fyn is localized to lipid rafts via tandem acylation and to caveolae through direct binding to caveolin. Upon activation, Fyn phosphorylates caveolin-1 on Tyr14. Tyrosine phosphorylation promotes SH2 domain-mediated binding of Abl. This in turn leads to the phosphorylation of adjacent caveolin molecules.

port this model, and give significant insights into the reason that caveolin phosphorylation by Fyn alone is self-limited and requires the activation of two non-receptor tyrosine kinases, Fyn and Abl, for sustained high level phosphorylation to occur.

Signaling Pathways Downstream of Caveolin Tyrosine Phosphorylation

With the exception of sites within the activation loops of kinases themselves, the function of tyrosine phosphorylation is to promote protein-protein interactions, particularly through SH2 protein-binding domains. This in turn leads to the activation of downstream signaling cascades. Therefore, phosphorylation of caveolin on tyrosine is likely to be an intermediate step in a signaling cascade occurring within caveolae. Caveolin-1 phosphorylated at Tyr14 would serve as a docking site for SH2 domain-containing proteins and would recruit proteins into caveolae to activate downstream signaling cascades (Fig. 6.3).

In order to identify proteins that bind to phosphorylated caveolin-1, a 3T3-L1 adipocyte cDNA library was screened using a novel yeast dihybrid screen [78]. The Gal4-based yeast two-hybrid system was modified to perform a phosphotyrosine-dependent dihybrid protein interaction screen. Then, a kinase (Abl) was introduced into the two-hybrid system to phosphorylate the bait protein (caveolin-1), after which screening was carried out for phosphorylation-dependent protein interactions. Using this system to screen an adipocyte cDNA library, three proteins were identified that interact with the amino-terminus of caveolin-1: JAB1, TRAF2, and Csk. Of these three proteins, only Csk contains an SH2 domain.

Fig. 6.3 Activation of signaling cascades downstream of caveolin phosphorylation. Extracellular signals (insulin and stress) activate caveolin kinases (Fyn and Abl). This leads to phosphorylation of caveolin-1 on

Tyr14. Tyrosine phosphorylation promotes SH2 domain-mediated protein interactions. This in turn leads to activation of downstream signaling cascades within the caveo-lae.

Fig. 6.3 Activation of signaling cascades downstream of caveolin phosphorylation. Extracellular signals (insulin and stress) activate caveolin kinases (Fyn and Abl). This leads to phosphorylation of caveolin-1 on

Tyr14. Tyrosine phosphorylation promotes SH2 domain-mediated protein interactions. This in turn leads to activation of downstream signaling cascades within the caveo-lae.

Csk Binds to Phosphocaveolin

Based on the initial screen, positive proteins could interact with caveolin-1 in a phosphorylation-dependent or independent manner. In order to counter-screen for phosphorylation-dependent interactions, the positive clones were transformed back into yeast that expressed caveolin-1 alone, caveolin-1 and Abl, or the phosphorylation mutant caveolin-1/Y14F and Abl. Only the interaction of Csk with caveolin-1 was completely dependent on phosphorylation. TRAF2 also bound to non-phosphorylated caveolin-1, although phosphorylation of caveolin-1 increased the binding of TRAF2 to caveolin-1 approximately three-fold. The proteasome subunit JAB1 interacted with caveolin-1 in a phosphorylation-independent manner.

Previous studies had shown that TRAF2 and caveolin-1 form a complex that recruits the tumor necrosis factor-alpha (TNFa) receptor after ligand binding [90]. Therefore, focus was centered on Csk. The interaction of Csk with phosphocaveolin in mammalian cells was verified. Phosphocaveolin co-immunoprecipitated with Csk in cells expressing v-Abl, and was one of only two major tyrosine-phos-phorylated proteins bound to Csk in these cells [78]. The other phosphoprotein was paxillin, a multi-domain focal adhesion protein known to bind to Csk through an SH2 domain-phosphotyrosine interaction. Phosphocaveolin also co-immunopreci-pitated with Csk in untransfected cells and this association increased after induction of oxidative stress or shear stress, and in adipocytes after stimulation with insulin [24,34,78,91]. Csk interacted only with caveolin phosphorylated on Tyr14, and did not bind to non-phosphorylated caveolin. Csk is one of only two proteins that are known to bind specifically to phosphorylated caveolin-1; the SH2 domain-

containing protein Grb7 is the other [16]. Only Csk has been shown to interact with caveolin-1 in a regulated manner in cells.

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