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|Title:||The mechanism of action of sprouty2: Characterization of the interaction between sprouty2 and PKCdelta||Authors:||CHOW SOAH YEE||Keywords:||Sprouty, PKCdelta, PKD1, ERK, cell invasion, FGFR1||Issue Date:||26-Jun-2009||Citation:||CHOW SOAH YEE (2009-06-26). The mechanism of action of sprouty2: Characterization of the interaction between sprouty2 and PKCdelta. ScholarBank@NUS Repository.||Abstract:||Sprouty (Spry) proteins function as inhibitors of receptor tyrosine kinase (RTK) signaling. Through their action, they play a crucial role in regulating branching morphorgenesis, development and cell migration. The best characterized function of Spry proteins is their role in inhibiting RTK-mediated ERK1/2 activation. In the past few years, studies by different groups have been carried out to elucidate the mechanisms by which Spry proteins are able to inhibit ERK activity. Most of the work performed to date concentrate on the canonical RTK-Grb2-Sos-Ras-Raf-MEK-ERK pathway. Current evidence suggests different points of action, including upstream of Ras and Raf.
In this study, the question was raised as to whether Spry proteins could influence other signaling pathways, and what impact they would have. Identification of novel interacting proteins using a yeast two-hybrid screen was employed as a strategy to answer this question. Phospholipid scramblase 3 (Plscr3) was isolated from this screen, and through this protein, protein kinase Cdelta (PKCdelta) was further identified to be a FGF stimulation-dependent interacting partner of Spry2. The interaction between Spry2 and PKCdelta was also found to be both specific and direct, and it depends on the conformation of the two proteins.
The binding of Spry2 and PKCdelta does not inhibit phosphorylation or activation of PKCdelta. Instead, it inhibits the phosphorylation of a PKCdelta substrate, protein kinase D1 (PKD1), on two serine residues within its activation loop. Further analysis showed that Spry2, PKCdelta and PKD1 form a trimeric complex. In order for Spry2 to interact with PKCdelta, PKD1 and PKCdelta must first bind to each other. The role that Spry2 plays within this complex is to lock the interaction between PKCdelta and PKD1, and block the transfer of a phosphate group from PKCdelta to PKD1. The interaction between Spry2 and PKCdelta therefore effectively creates a kinase-dead PKCdelta.
In this study, the kinase activity of PKCdelta is demonstrated to be required for ERK1/2 phosphorylation. Therefore, by inhibiting the ability of PKCdelta to phosphorylate its substrates, Spry2 is able to limit the contribution of PKCdelta signaling to ERK1/2 activation. This takes place through the absence of phosphorylation of PKD1, which is necessary for its activation. It is proposed that its substrate, Ras inhibitor 1 (RIN1), is maintained in an unphosphorylated state, which acts as an effective competitor of Raf for Ras binding. Signal propagation between Ras and Raf would therefore be reduced. Results from this study indicate that the expression of Spry2 increases the interaction between active Ras and RIN1.
Reports have suggested that the kinase activity of PKCdelta is required for the invasive potential of prostate cancer cells. Cell invasion assays in this study show that by inhibiting phosphorylation of its substrates, Spry2 is able to block PKCdelta-mediated cell invasion.
The results from this study indicate that Spry2 is able to regulate a pathway that is distinct from the canonical Ras/ERK signaling pathway. Furthermore, it is demonstrated that this pathway contributes to ERK phosphorylation. The interaction between Spry2 and PKCdelta, a component of this pathway, represents a novel method in which Spry2 is able to inhibit ERK1/2 activation.
|Appears in Collections:||Ph.D Theses (Open)|
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