Insights into molecular interactions between the juxtamembrane and kinase subdomains of the Arabidopsis Crinkly-4 receptor-like kinase

• Published in: Archives of biochemistry and biophysics 2013-07, Vol.535 (2), p.101-110
• Main Authors: Meyer, Matthew R., Shah, Shweta, Rao, A. Gururaj
• Format: Article
• Language: English
• Subjects: Amino Acid Motifs; Amino Acid Sequence; Arabidopsis; Arabidopsis Proteins - chemistry; Arabidopsis Proteins - genetics; bacteriophages; Consensus Sequence; growth and development; Intramolecular interaction; Juxtamembrane domain; Kinetics; Models, Molecular; Molecular Sequence Data; mutagenesis; Mutagenesis, Site-Directed; mutants; peptide libraries; Peptide Library; Peptides - chemistry; Phage-peptide library; Phosphorylation; plant growth; Protein Binding; Protein Structure, Tertiary; protein-protein interactions; Protein-Serine-Threonine Kinases - chemistry; Protein-Serine-Threonine Kinases - genetics; proteins; Receptor-like kinase; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - genetics; screening
• ISSN: 0003-9861; 1096-0384
• DOI: 10.1016/j.abb.2013.03.014

[Display omitted] •The juxtamembrane domain (JMD) of the receptor kinase ACR4 contains 2 phosphosites.•Naïve and phosphorylated JMD peptides were panned against a phage-peptide library.•The consensus sequence LxSLL is recognized by the phosphorylated (pJMD) peptides.•pJMD peptides bind specifically to ACR4 kinase domain containing the LxSLL motif.•An intramolecular interaction is suggested between the JM and kinase domain of ACR4. Arabidopsis CRINKLY4 (ACR4), a receptor-like kinase required for plant growth and development, possesses an extracellular ligand binding domain, a transmembrane helix, and an intracellular domain (ICD). The ICD contains the juxtamembrane (JMD) and the C-terminal (CTD) subdomains, which flank the core kinase domain (KD), with at least 16 autophosphorylation sites. Phosphorylation sites are often docking sites for the modification-dependent recruitment of interacting proteins that orchestrate many downstream signaling events. In this context, we have specifically probed the role of the two phosphorylation sites Ser475 and Thr478 in the JMD using mutagenesis and phage-peptide screening techniques. Thus, naïve and phosphorylated 15-mer peptides derived from the JMD were panned against a 21-amino acid random phage peptide library. The phosphorylated peptide preferentially recognized the consensus sequence LxSLL. This sequence harbors the LxxLL motif, a known protein–protein interaction motif that is also present in the N-terminal lobe of the KD. We demonstrate the binding of JMD peptides to the KD and also show through kinetic analyses of mutants that phosphorylation of Ser475 and Thr478 in the JMD is necessary for optimal substrate phosphorylation in vitro. Our experiments suggest that an intramolecular interaction can occur between the JM and the N-terminal lobe of the KD.