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.

14Small RHO-type G-proteins act as signaling hubs and master regulators of polarity in 15 eukaryotic cells. Their activity is tightly controlled, as defective RHO signaling leads to aberrant 16 growth and developmental defects. Two major pathways regulate G-protein activity: canonical 17 switching of the nucleotide bound state and posttranslational modification (PTM). PTMs can 18 support or suppress RHO signaling, depending on each individual case. In plants, regulation 19 of Rho of plants (ROPs) has been shown to act through nucleotide exchange and hydrolysis, 20 as well as through lipid modification, but there is little data available on phosphorylation or 21 ubiquitination of ROPs. Hence, we applied proteomic analyses to identify PTMs of the barley 22 ROP RACB. We observed in vitro phosphorylation by barley ROP Binding Kinase 1 and in vivo 23 ubiquitination of RACB. Comparative analyses of the newly identified RACB phosphosites and 24Rac3, we suggest that RHO family proteins from different kingdoms could be generally 29 regulated by ubiquitination of this site. 30 31Lipidations are not the only posttranslational modifications (PTM) in RHO proteins. In animals 58 both phosphorylation and ubiquitination are employed to control RHO signaling, thereby 59 enhancing or suppressing G-protein function in a case-dependent manner. The mammalian 60 RHO family member Rac1 for instance is phosphorylated at three amino acids by different 61 kinases: focal adhesion kinase (FAK) and proto-oncogene tyrosine-protein kinase Src (cellular 62 sarcoma) phosphorylate Rac1 in vitro at Y64 (15). Phosphomimetic Rac1-Y64D mutants 63 showed decreased GTP-binding and association with downstream signaling proteins, and cells 64 expressing Rac1-Y64D displayed defects in focal adhesion targeting and cell spreading. 65Extracellular signal-related kinase (ERK) phosphorylates Rac1 at T108, targeting it to the 66 nucleus before prenylation and hence isolating it from activation by cytosolic GEFs (16). 67 Phosphorylation of Rac1 by the AGC kinase AKT (RAC-alpha serine/threonine-protein kinase 68 1) at S71 has been shown to be essential for targeting by an F-box/Leucine-rich repeat (FBXL) 69 protein (17, 18). F-box proteins are part of SKP1-CUL1-F-box (SCF) E3 ubiquitin ligase 70 complexes which mediate ubiquitination and proteasomal degradation of selected proteins 71 (19). SCF FBXL19 targets S71-phosphorylated Rac1, which leads to ubiquitination of Rac1 K166 72 (18). This depletes Rac1 from cells, which inhibits its function in cell spreading. Another Rac1 73 ubiquitination site is K147, which is targeted by HECT domain and ankyrin repeat containing 74 E3 ubiquitin protein ligase 1 (HACE1) and two inhibitor of apoptosis proteins (IAPs). While 75 HACE1 mainly targeted GTP-bound Rac1, both XIAP and c-IAP1 did not show any preference 76 for a particular nucleotide-bound state. In all cases, ubiquitination of K147 leads to proteasomal 77 degradation of Rac1, abolishing its function in cell migration (20-22). For mammalian RhoA, 78 four phosphorylat...