In silico identification and experimental validation of amino acid motifs required for the Rho-of-plants GTPase-mediated activation of receptor-like cytoplasmic kinases

Abstract

Several amino acid motifs required for Rop-dependent activity were found to form a common surface on RLCKVI_A kinases. This indicates a unique mechanism for Rho-type GTPase-mediated kinase activation in plants. Rho-of-plants (Rop) G-proteins are implicated in the regulation of various cellular processes, including cell growth, cell polarity, hormonal and pathogen responses. Our knowledge about the signalling pathways downstream of Rops is continuously increasing. However, there are still substantial gaps in this knowledge. One reason for this is that these pathways are considerably different from those described for yeast and/or animal Rho-type GTPases. Among others, plants lack all Rho/Rac/Cdc42-activated kinase families. Only a small group of plant-specific receptor-like cytoplasmic kinases (RLCK VI_A) has been shown to exhibit Rop-binding-dependent in vitro activity. These kinases do not carry any known GTPase-binding motifs. Based on the sequence comparison of the Rop-activated RLCK VI_A and the closely related but constitutively active RLCK VI_B kinases, several distinguishing amino acid residues/motifs were identified. All but one of these were found to be required for the Rop-mediated regulation of the in vitro activity of two RLCK VI_A kinases. Structural modelling indicated that these motifs might form a common Rop-binding surface. Based on in silico data mining, kinases that have the identified Rop-binding motifs are present in Embryophyta but not in unicellular green algae. It can, therefore, be supposed that Rops recruited these plant-specific kinases for signalling at an early stage of land plant evolution.

The Arabidopsis Rho of Plants GTPase ROP1 Is a Potential Calcium-Dependent Protein Kinase (CDPK) Substrate

“…However, much less is known about the link of the related plant-specific ROP G-proteins to kinases since none of the Rho-interacting kinase families that were described in animals exist in plants [12]. As kinases downstream of ROPs are considered, the members of the Arabidopsis Receptor-like Cytoplasmic Kinase Class VI GroupA (RLCK VI_A) and related proteins from other species are good candidates as direct ROP signalling partners [44][45][46][47]. As the upstream regulation of ROPs by kinases is considered, it is well-accepted that several plant receptor-like kinases (RLKs) control ROP GTPase signalling through RopGEFs, the activators of ROPs [12,48].…”

The Arabidopsis RLCK VI_A2 Kinase Controls Seedling and Plant Growth in Parallel with Gibberellin

“…However, plant genomes code for none of these kinases [ 11 ]. It seems that, during the evolution of land plants, a sub-group of plant-specific RLCKs were co-opted to mediate ROP GTPase signalling [ 12 ]. While the GTPase-binding ability of yeast and animal Rho-type GTPase-regulated kinases is due to the presence of defined structural elements outside of their kinase domains (such as the Cdc42/Rac-interactive binding -CRIB- motif of PAKs), RLCK VI_A kinases use conserved amino acids widely distributed in the kinase domain to form a binding surface for ROPs [ 12 ].…”

The Effects of Exogenous Polyamines and the Silencing of RLCK VI_A Kinases on the Polar Growth of Nicotiana tabacum (Tobacco) Pollen Tubes

“…The ROPs regulate multiple extracellular signals such as cell growth, development, cytoskeleton dynamics/reorganization and vesicular trafficking required for polarity establishment/ maintenance (including pollen tube tip growth), reproduction, and responses to the environment (Feiguelman et al 2018).Their function is exerted via the spatial and temporal activation of various downstream effector proteins (Feiguelman et al 2018). Among these, RLCK VI_A kinases are the only known potential ROP effector kinases (Dorjgotov et al 2009;Lajkó et al 2018), but their link to ROP-mediated signalling pathways could not be demonstrated yet in planta.…”

“…The first group RLCK VI_A (characterized by N-terminal serine-rich region) contains ROP-interacting and the second group RLCK VI_B (characterized by N-terminal UspA domain, Kerk et al 2003) ROP-non-interacting kinases (Jurca et al 2008). Members of the RLCK VI_A group are the only currently known plant kinases the activity of which is dependent on their binding to active (GTP-bound) ROP G-proteins (Dorjgotov et al 2009;Lajkó et al 2018). Besides the ROP-dependent regulation of their activity, there is information about the expression of the members of this family in Arabidopsis thaliana.…”

“…Although there are reports about the use of the AS-ODN technology for Arabidopsis pollen tubes (Mizuta and Higashiyama 2014), we had difficulties to reproducibly germinate and grow Arabidopsis pollens. Nicotiana tabacum pollen, however, can easily be collected and cultured and were routinely used in our laboratory for gain-of-function studies (Fodor-Dunai et al 2011;Lajkó et al 2018). Therefore, members of the RLCK VI_A family of Nicotiana tabacum were identified based on their homology to the Arabidopsis sequences.…”