Minisymposium 12: Intracellular Signaling

Abs # 24001: An Arabidopsis protein with sequence similarity to animal TGF-beta Receptor Interacting Protein is phosphorylated by the BRI1 receptor kinase in vitro

Presenter:	Ehsan, Hashimul , hehsan@unity.ncsu.edu
Authors	Ehsan, Hashimul  (A)   Ray, William K. (B)   Huber, Steven C. (A)   Clouse, Steven D. (A)
Affiliations:	(A): North Carolina State University (B): Michigan State University

Brassinosteroids (BRs) are growth-promoting natural products found at low levels in pollen, seeds and young vegetative tissues throughout the plant kingdom. The analysis of BR-insensitive and BR-deficient mutants has shown that these steroids are essential signals controlling plant growth and development. BRs regulate the expression of numerous genes associated with plant development and require the activity of BRI1, a Ser/Thr receptor kinase, to realize their effects. The Transforming Growth Factor-beta (TGF-beta) family of peptides, acts via Ser/Thr receptor kinases to prominently impact several pathways involved in animal development and adult homeostasis. In animals, TGF-beta Receptor Interacting Protein (TRIP-1) is an intracellular substrate of the TGF-beta Type II receptor kinase and plays an important role in TGF-beta signaling. TRIP-1 is a WD-repeat protein that also has a dual role as an essential subunit of the eukaryotic translation initiation factor eIF3 in animals, yeast and plants, thereby revealing a putative link between a developmental signaling pathway and the control of protein translation. We recently found that transcript levels of TRIP-1 homologs in plants are regulated by BR treatment under a variety of conditions, and that transgenic plants expressing antisense TRIP-1 RNA exhibit a broad range of developmental defects including some that resemble the phenotype of BR-deficient and -insensitive mutants. Furthermore, we have shown that recombinant BRI1 kinase domain phosphorylates recombinant TRIP-1 in vitro. These findings suggest that TRIP-1 may mediate some of the molecular mechanisms underlying the regulation of plant growth and development by BRs, possibly as a direct substrate of the BRI1 receptor kinase. We have identified three Thr residues in TRIP-1 that are the putative targets of BRI1 phosphorylation, using MALDI and Q-TOF mass spectrometry. Immunoprecipitation followed by mass spectrometry is also being used to identify in vivo phosphorylation sites of TRIP-1 and their possible dependence on an active BRI1 receptor kinase. Recent co-immunoprecipitation experiments using antibodies against TRIP-1, BRI1 and various fusion proteins strongly suggest that TRIP-1 and BRI1 interact in vivo, further strengthening the argument that TRIP-1 is a substrate of the BRI1 kinase domain.