Receptor kinases are essential to perceive the signals that induce cellular events such as cell division, cell differentiation and cell death. The Somatic Embryogenesis Receptor-like Kinase 1 ( At SERK1) is a receptor-like kinase (RLK) containing five leucine-rich repeats in its extracellular domain. At SERK1 is proposed to participate in a signal transduction cascade involved in ovule and embryo development. The downstream components of the At SERK1 signal transduction cascade are mostly unknown. Therefore we used the yeast two-hybrid system to screen for proteins interacting with the kinase domain of At SERK1. As a result of the screen we found six different putative At SERK1 interacting proteins. These proteins were predicted to encode a member of the Arabidopsisthaliana family of 14-3-3 proteins called GF14l, the AAA-ATPase At CDC48, a putative RNA binding protein (RBP), the potassium transporter KEA1, the aquaporin At SIP1 and a racemase-like protein. As we were looking for proteins that may be involved in the signal transduction cascade mediated by the At SERK1 receptor kinase we performed i n vitro kinase assays to determine which proteins At SERK1 can use as a substrate. GF14l, At CDC48 and RBP can be transphosphorylated by At SERK1 in vitro . Therefore, only the interaction of At SERK1 with these proteins was studied in further detail.
In vitro binding assays were performed to confirm the interaction with At SERK1 and showed that GF14lpreferably binds to the phosphorylated At SERK1 kinase domain. The interaction between At SERK1 and GF14lin vivo was demonstrated by co-immunoprecipitation assays and in plant cells by the occurrence of FRET between fluorescently labelled At SERK1 and GF14lproteins. In the kinase domain of At SERK1 we found a putative 14-3-3 binding motif with the sequence RPPSQPP surrounding Ser-394. This motif is located in a loop opposite to the active site of the kinase molecule. Beside At SERK1 another 20 Arabidopsis receptor-like kinases contain a putative 14-3-3 binding domain in this loop.
Like GF14l, the ATPase At CDC48 preferably binds to the phosphorylated kinase domain in an in vitro binding assay and the in vivo interaction was demonstrated by co-immunoprecipitation assays. Upon binding, At SERK1 is able to transphosphorylate At CDC48 on a threonine residue within the C-terminal 62 residues of the protein. In yeast, At CDC48 is also able to interact with the At SERK1-binding partners GF14land the protein phosphatase KAPP. The interaction between At CDC48 and a 14-3-3 protein was confirmed in plant cells by co-immunoprecipitation. This shows analogy to the mammalian CDC48 homologue p97/VCP, which can be phosphorylated by the JAK-2 kinase and dephosphorylated by the phosphatase PTPH1 that associates with a 14-3-3 protein. Based on the function and the cellular localisation of At CDC48 we propose that At SERK1 is involved in one of the many controlling pathways of the cell cycle. The observation that several Arabidopsis protein kinases predicted to be involved in cell division also contain a putative 14-3-3 binding sequence supports this hypothesis.
|Qualification||Doctor of Philosophy|
|Award date||2 Jun 2003|
|Place of Publication||[S.I.]|
|Publication status||Published - 2003|
- arabidopsis thaliana
- somatic embryogenesis
- protein kinase
- signal transduction
- cell interactions