Posttranslational modification of the RHO of plants protein RACB by phosphorylation and cross-kingdom conserved ubiquitination

Abstract

Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant growth and developmental defects. Two major processes regulate G-protein activity: canonical shuttling between different nucleotide bound states and posttranslational modification (PTM), of which the latter can support or suppress RHO signaling, depending on the individual PTM. In plants, regulation of Rho of plants (ROPs) signaling activity has been shown to act through nucleotide exchange and GTP hydrolysis, as well as through lipid modification, but there is little data available on phosphorylation or ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley ROP RACB. We observed in vitro phosphorylation by barley ROP binding kinase 1 and in vivo ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and human RHO protein phosphosites revealed conservation of modified amino acid residues, but no overlap of actual phosphorylation patterns. However, the identified RACB ubiquitination site is conserved in all ROPs from Hordeum vulgare, Arabidopsis thaliana and Oryza sativa and in mammalian Rac1 and Rac3. Point mutation of this ubiquitination site leads to stabilization of RACB. Hence, this highly conserved lysine residue may regulate protein stability across different kingdoms.

Barley RIC157, a potential RACB scaffold protein, is involved in susceptibility to powdery mildew

“…In contrast to that, a strong re-localization of GFP-RIC157 to the cell periphery was observed in the presence of mCherry-tagged CARACB(G15V) that likewise accumulated at this site. RACB, like other ROPs associates with the plasma membrane probably via its C-terminal posttranslational lipidations (Schultheiss et al 2003 ; Weiß et al 2022 ). Activated RACB then apparently recruits RIC157 to the cell periphery.…”

“…Transient silencing of RBK1 or RBK1-interacting protein SKP1 (type II S-phase kinase1-associated protein) suggested that RBK1 negatively regulates RACB protein stability and hence rather acts in disease resistance (Reiner et al 2016). A highly conserved lysine residue that is targeted for ubiquitination further supports the idea of proteasomal regulation of RACB (Weiß et al 2022).…”

“…This supports the hypotheses that RACB activation precedes the recruitment of interaction partners and downstream RACB signaling is initiated at the plasma membrane. The latter point is further backed up by a cytoplasm localised RACB mutant version lacking the C-terminal CSIL motif required for prenylation that is inactive in promoting susceptibility to fungal penetration (Schultheiss et al 2003 ; Weiß et al 2022 ).…”

Common Post-translational Modifications (PTMs) of Proteins: Analysis by Up-to-Date Analytical Techniques with an Emphasis on Barley

“…This indicates that the lysine residue belonging to RACB K167 is a general target for ubiquitination and that this regulates the amount of protein. 90 SUMOylation is a key player in plant immunity and, thus, plays a positive role in fungal resistance. By studying the molecules accumulated in barley, a certain level of resistance to Fusarium graminearum was confirmed, leading to the identification of resveratrol, a candidate inhibitor of SUMO proteases.…”

“…It was found that the ubiquitination site is maintained in all barley ROPs. This indicates that the lysine residue belonging to RACB K167 is a general target for ubiquitination and that this regulates the amount of protein …”