| Arabidopsis CRINKLY4 (ACR4) is a receptor-like kinase (RLK) that is required for the global development of the plant. In aerial tissues ACR4 promotes the differentiation of the outer, epidermal layer in the leaves and reproductive tissues. Loss of ACR4 function results in malformed, 'crinkled' leaves due to graft-like fusions of vegetative tissues. Underground, ACR4 is responsible for stem cell differentiation in the root tip and for the initiation of lateral roots. Acr4 mutant plants show increased proliferation of stem cells and increase lateral root initiation sites. ACR4 has architectural features analogous to receptor tyrosine kinases (RTK). It includes an extracellular ligand binding domain, a transmembrane helix, and an intracellular kinase domain. The intracellular domain contains two subdomains, the juxtamembrane domain (JMD) and the C-terminal domain (CTD) which flank the core kinase domain (KD). The Arabidopsis genome also encodes four homologs of ACR4, referred to as Arabidopsis CRINKLY4 Related (AtCRR) proteins, that contain equivalent architectural domains to ACR4 and have high sequence similarity. Genetic and cell biology studies have suggested potential communication between members of the ACR4 gene family. However, a biochemical basis for receptor hetero-oligomerization among these family members has not been established. An additional RLK, ABNORMAL LEAF SHAPE 2 (ALE2), is involved in proper epidermal layer formation. ALE2 is also known to synergistically enhance ACR4 kinase activity in vitro . Furthermore, the spatial expression of a transcription factor, WOX5, has been demonstrated to be regulated by ACR4 signaling. However, the precise signaling pathway(s) modulated by these RLKs and the molecular basis of their biological function remain to be determined.;Although significant advancements have been made in understanding the biological relevance of ACR4, little is known about the biochemical aspects of ACR4 signaling. In this dissertation, we provide a fundamental biochemical analysis of the activation of the ACR4 intracellular kinase domain. We have expressed and purified the intracellular domains of ACR4, the AtCRRs, and ALE2 as C-terminal fusions to the yeast SUMO protein. We provide evidence for an intramolecular interaction mechanism between the ACR4 JMD and the amino-terminal region of the KD and demonstrate its potential role in the regulation of kinase activity. Additionally, we provide evidence for an intermolecular interaction mechanism between ACR4 and the AtCRRs, and potential regulation of this interaction by the ACR4 JMD. Lastly, we demonstrate that ACR4 can bind to the WOX5 transcription factor and phosphorylate it in vitro at four sites. |
