RESUMO
CRINKLY4 (CR4) is a plant serine-threonine receptor kinase. In Zea mays, CR4 functions in the differentiation of the leaf epidermis and the aleurone cell layer and, in Arabidopsis thaliana, the ortholog ACR4 is involved in the development of the integument and seed coat. The Arabidopsis genome also encodes four CR4-related proteins (CRR) whose functions are not known. Based on studies of animal receptor kinase proteins it is likely that the molecular basis of function of CR4 and related proteins is mediated by receptor dimerization. The importance of the transmembrane (TM) domain in the dimerization of several receptor kinases has been demonstrated by the TOXCAT system, a genetic assay that measures helix interactions in a natural membrane environment. In this study, we have used the TOXCAT assay to investigate the potential of the CR4 and CR4-related TM domains to homo-dimerize. Our investigation indicates that the CR4 TM domain and the CRR TM domains have higher propensities for homo-dimerization than the ACR4 TM domain. Interestingly, the dimerization potential of the ACR4 TM domain is significantly weaker even though 13 of 24 amino acids are identical to that of the CR4 TM domain. In order to determine the contributions of specific amino acids to the higher dimerization potential of CR4 compared to ACR4, mutations were made at specific sites in ACR4 TM domain and the strength of the dimer assessed by the TOXCAT assay. One mutation restored the activity to the CR4 level, while other mutations produced either no change or significantly increased the dimerization potential of the ACR4 TM domain. Our results indicate that the TM domains of CR4, ACR4 and the CRR receptor family of proteins have the intrinsic capacity to homo-dimerize, albeit with varying degrees of affinity.
Assuntos
Arabidopsis/enzimologia , Membrana Celular/química , Escherichia coli/enzimologia , Microdomínios da Membrana/química , Proteínas de Plantas/química , Proteínas Quinases/química , Sítios de Ligação , Dimerização , Isoenzimas/química , Ligação ProteicaRESUMO
The optimal kinase activity of plant receptor-like kinases (RLKs) is often regulated by autophosphorylation of specific sites. Many of these phosphorylated residues then serve as recruiting sites for downstream interacting proteins. Therefore, identification of the phosphosites can be an important first step in delineating the signaling network. This chapter describes a protocol for identification of phosphorylated residues by mass spectrometry as well as a protocol to determine if an interacting partner can be phosphorylated in vitro.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Mapeamento de Interação de Proteínas/métodos , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/genética , Receptores de Superfície Celular/genética , Sequência de Aminoácidos , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Sítios de Ligação , Cromatografia de Afinidade/métodos , Cromatografia Líquida/métodos , Expressão Gênica , Isoenzimas/genética , Isoenzimas/metabolismo , Mutagênese Sítio-Dirigida , Fosforilação , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de Superfície Celular/metabolismo , Espectrometria de Massas em Tandem/métodosRESUMO
A method is described for isolating the high mol. wt. protein fraction of mustard and rapeseed in a homogeneous form. The protein from the two species had nearly identical sedimentation coefficients, molecular weights, intrinsic viscosity and fluorescence emission spectrum. However, differences in the amino acid composition were observed. Data on circular dichroism and rate of hydrolysis by proteolytic enzymes suggested differences in the secondary structures and possibly amino acids sequence.