Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain

Authors: OH, MAN-HO - University Of Illinois; CLOUSE, STEVEN - North Carolina State University; Huber, Steven

Submitted to: Frontiers in Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/15/2012
Publication Date: 8/8/2012
Citation: Oh, M., Clouse, S.D., Huber, S.C. 2012. Tyrosine phosphorylation of the BRI1 receptor kinase occurs via a posttranslational modification and is activated by the juxtamembrane domain. Frontiers in Plant Physiology. 3:175.

Interpretive Summary: Many aspects of plant growth and development are controlled by proteins known as receptor kinases that are embedded in the outer cell membrane and transmit signals from the outside of the cell to the nucleus resulting in changes in gene expression. The receptor kinases become active when the specific signal they recognize is present. One of the best studied examples is the receptor kinase known as BRI1 that responds to plant steroid hormones and increases plant growth. When the steroid hormones are present, the protein kinase activity of the receptor becomes activated when it attaches phosphate molecules to itself in a process known as autophosphorylation. Once activated in this manner, the BRI1 protein kinase can phosphorylate (attach phosphate molecules to) other proteins and this way the signal is transmitted ultimately to the nucleus. The ability of BRI1 to autophosphorylate on tyrosine residues has only recently been demonstrated and it was not clear whether the autophosphorylation occurred while the BRI1 protein was being formed (referred to as a co-translational mechanism) or whether the process occurred after the protein was fully formed (referred to as a post-translational modification). These two possibilities are important to distinguish because each would predict different regulatory strategies. The present study shows that the process of autophosphorylation involves mature BRI1 protein molecules and is readily reversible. Moreover, new results also show that regions of the molecule flanking the protein kinase domain affect the ability to phosphorylate on tyrosine residues compared to serine and threonine residues. This was surprising because the protein kinase domain is generally considered autonomous in terms of specificity. These findings provide new insights into the molecular mechanisms that control a receptor kinase that plays a major role in control of growth of crop plants and may ultimately yield new strategies for increasing productivity.

Technical Abstract: In metazoans, receptor kinases control many essential processes related to growth and development and response to the environment. The receptor kinases in plants and animals are structurally similar but evolutionarily distinct from one another, and thus while most animal receptor kinases are tyrosine kinases the plant receptor kinases are classified as serine/threonine kinases. One of the best studied plant receptor kinases is BRASSINOSTEROID INSENSITIVE 1 (BRI1), which functions in brassinosteroid (BR) signaling. Consistent with its classification, BRI1 was shown in early studies to autophosphorylate in vitro exclusively on serine and threonine residues and subsequently numerous specific phosphoserine and phosphothreonine sites were identified. However, several sites of tyrosine autophosphorylation have recently been identified establishing that BRI1 is a dual-specificity kinase. This raises the paradox that BRI1 contains phosphotyrosine but was only observed to autophosphorylate on serine and threonine sites. In the present study, we demonstrate that autophosphorylation on threonine and tyrosine (and presumably serine) residues is a post-translational modification, ruling out a co-translational mechanism that could explain the paradox. Moreover, we show that in general, autophosphorylation of the recombinant protein appears to be hierarchal and proceeds in the order: phosphoserine > phosphothreonine > phosphotyrosine. We suggest that this may be one factor that explains why tyrosine autophosphorylation was not observed in earlier autophosphorylation studies. Finally, we also confirm that the juxtamembrane domain of BRI1 is an activator of the kinase domain, and is essential for autophosphorylation on threonine and tyrosine residues. Furthermore, analysis of a nested series of juxtamembrane truncation mutants, and alanine-scanning mutants of selected juxtamembrane sequences, demonstrates that kinase specificity (serine/threonine versus tyrosine) can be affected by residues outside of the kinase domain. This may have implications for identification of signature motifs that distinguish serine/threonine kinases from dual-specificity kinases.