Concluding Remarks

The pace of recent discoveries in BR signaling has been rapid and informative, as summarized in Fig. 4. Only 3 years ago several unanswered but critical questions regarding BR signaling could be posed, such as:

1. Do BRI and BAK1 form true co-receptors in vivo?

2. Are accessory steroid-binding proteins required for BR binding to BRI1 and/or BAK1?

3. What are the in vivo autophosphorylation sites of BRI1 and BAK1, and what are their BR dependencies?

4. What are the number and nature of cytoplasmic binding partners of the BRI1 and BAK1 kinase domains?

5. What is the mechanism of BR-dependent inactivation of BIN2 kinase activity?

6. How do BES and BZR1 participate in the regulation of specific genes?

Fig. 4 Summary of components and interactions in BR signaling. See text for discussion of each step in the pathway. The nucleus is not shown due to some uncertainty in the localization of some downstream components. BR-induced transport of hypophospho-rylated BES1/BZR1 from the cytoplasm to the nucleus has been proposed as has, more recently, constitutive localization of BES1/BZR1 in the nucleus where phosphorylation and dephosphorylation occur through the action of BIN2 kinase and BSU1 phosphatase

Fig. 4 Summary of components and interactions in BR signaling. See text for discussion of each step in the pathway. The nucleus is not shown due to some uncertainty in the localization of some downstream components. BR-induced transport of hypophospho-rylated BES1/BZR1 from the cytoplasm to the nucleus has been proposed as has, more recently, constitutive localization of BES1/BZR1 in the nucleus where phosphorylation and dephosphorylation occur through the action of BIN2 kinase and BSU1 phosphatase

Biochemical approaches, such as immunoprecipitation, LC/MS/MS analysis, FLIM and FRET localization studies, and photo-affinity labeling have clearly shown that BL binds directly to the extracellular domain of BRI1, that BRI1 and BAK1 interact in a ligand-dependent manner and are phosphory-lated on specific Ser and Thr residues in response to BR. Detailed analysis of BES and BZR1 action has shown that the hypophosphorylated forms of these novel transcription factors bind to specific BR responses elements. Extensive microarray studies have revealed hundreds of BR-regulated genes for further study. Both genetic screens and biochemical approaches have been useful in characterizing three initial substrates of the BRI1 kinase domain. However, a major gap in our understanding of BR signal transduction lies in the events that follow BRI1/BAK1 heterodimerization and phosphorylation and precede inactivation of the BIN2 kinase. Targets of BRI1/BAK1 phosphorylation that propagate the signal downstream need to be identified and approaches employing proteomic techniques, including LC/MS/MS analysis coupled with isotope coded affinity tagging procedures, may be the most global approach to identifying the full spectrum of BRI1 and BAK1 cytoplasmic substrates.

Acknowledgements Work in the author's laboratory is supported by the US National Science Foundation (MCB-0419819), the US Department of Agriculture (NRI 2004-3530414930), and the North Carolina Agricultural Research Service.

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