BRI1 Substrates and Interacting Proteins

Studies addressing the number and nature of cytoplasmic binding partners of the BRI1 and BAK1 kinase domains that propagate the signal downstream are essential for a complete understanding of BR action. Yeast two-hybrid screens were used to identify two probable in vivo substrates of BRI1 in Arabidopsis. TRANSTHYRETIN-LIKE protein (TTL) has substantial sequence identity to the vertebrate thyroid-binding protein transthyretin and interacts with BRI1 in yeast cells in a kinase-dependent manner (Nam and Li 2004). TTL is phos-phorylated by recombinant BRI1 kinase domain in vitro and overexpression of the TTL gene results in a semi-dwarf phenotype similar to weak bril and null bakl mutants, while null mutants of TTL enhance BR sensitivity and promote plant growth. Thus, genetic evidence suggests that TTL is a negative regulator of BR signaling, while in vitro evidence supports a role for TTL as a putative BRI1 substrate, although a direct interaction of the two proteins in planta has not been demonstrated (Nam and Li 2004).

A second interactor from the yeast two-hybrid screen, BKI1 (BRI1 KINASE INHIBITOR 1), is also a negative regulator of BR signaling. It is membrane-associated in the absence of BR and binds to BRI1, presumably inactivating some aspect of its function (Wang and Chory 2006). BR treatment causes dissociation of BKI1 from the membrane, which releases repression of the BR signaling pathway. Tethering BKI1 to the membrane by addition of a myris-toylation site results in an enhanced BR dwarf phenotype. BRI1 and BKI1 interact directly in vitro and in planta and BKI1 is phosphorylated by recombinant BRI1 kinase domain in vitro and is a phosphoprotein in vivo. The favored model for BKI1 action suggests that membrane-associated BKI1 binds directly to the kinase domain of BRI1, preventing its association with BAK1. Binding of BL to the extracellular domain of BRI1 activates the kinase domain, leading to dissociation of BKI1 from the membrane and allowing BRI1 and BAK1 to heterodimerize to initiate BR signaling (Wang and Chory 2006).

The third putative cytoplasmic substrate of BRI1 is the plant ortholog of mammalian TRIP-1. LC/MS/MS approaches show that recombinant Arabidopsis TRIP-1 is strongly phosphorylated by the BRI kinase domain in vitro, predominately on three specific residues (Thr-14, Thr-89, and either Thr-197 or Ser-198). Moreover, BRI1 and TRIP-1 co-immunoprecipitate from Arabidopsis plant extracts using native antibodies or various combinations of tagged proteins, suggesting an in vivo interaction between the two proteins (Ehsan et al. 2005). Some of the morphological characteristics of transgenic lines expressing antisense TRIP-1 RNA are also consistent with a possible role for TRIP-1 in BR signaling, although this in itself is not definitive proof (Jiang and Clouse 2001). Thus, TRIP-1 is a likely candidate for a BRI1 cytoplasmic substrate, but its functional role in BR signaling remains unclear. TRIP-1 (also known as eIF3i) is a dual function protein that has also been shown to be an essential subunit of the eIF3 translation initiation complex in mammals, yeast, and plants (Asano et al. 1997; Burks et al. 2001). This finding raises the intriguing possibility that BR-dependent phosphorylation of TRIP-1 by BRI1 may affect eIF3 activity and/or assembly and thus impact the global cellular phenomenon of protein translation, providing a novel mechanism for BR regulation of plant growth.

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