Abstract
Plant Rho-type GTPases (ROPs) are versatile molecular switches involved in a number of signal transduction pathways. Although it is well known that they are indirectly linked to protein kinases, our knowledge about their direct functional interaction with upstream or downstream protein kinases is scarce. It is reasonable to suppose that similarly to their animal counterparts, ROPs might also be regulated by phosphorylation. There is only, however, very limited experimental evidence to support this view. Here, we present the analysis of two potential phosphorylation sites of AtROP1 and two types of potential ROP-kinases. The S74 site of AtROP1 has been previously shown to potentially regulate AtROP1 activation dependent on its phosphorylation state. However, the kinase phosphorylating this evolutionarily conserved site could not be identified: we show here that despite of the appropriate phosphorylation site consensus sequences around S74 neither the selected AGC nor CPK kinases phosphorylate S74 of AtROP1 in vitro. However, we identified several phosphorylation sites other than S74 for the CPK17 and 34 kinases in AtROP1. One of these sites, S97, was tested for biological relevance. Although the mutation of S97 to alanine (which cannot be phosphorylated) or glutamic acid (which mimics phosphorylation) somewhat altered the protein interaction strength of AtROP1 in yeast cells, the mutant proteins did not modify pollen tube growth in an in vivo test.
Highlights
We have previously demonstrated that the S74E phosphorylation-mimic mutation of the Medicago sativa ROP6 protein interfered with the regulation and with the in vitro as well as in planta function of the GTPase [28]
The AGC1.7 kinase was selected based on its proven role in the polar growth of pollen tubes controlled by the ROP1 GTPase [31,32]
Since the conformation of ROP1 is dependent on nucleotide-binding that may affect its phosphorylation, dominant negative (DN with the T20N mutation locking the protein in the GDP-bound state) and constitutive active (CA with the G15V mutation resulting in the GTP-bound conformation) proteins were used as substrates apart from the wild-type one (WT)
Summary
Rho family GTPases are implicated in a wide range of basic cellular processes including cell morphology, polarity, motility, division, and defence [1]. The specificity of the PRONE domain towards ROPs is mainly due to two plant-specific amino acid residues, asparagine 68 (N68) and arginine 76 (R76) (based on Arabidopsis ROP4 numbering; Figure 1), close to the “switch II” region [17], involved in the GTP-bindingdependent conformation switch of G-proteins [18] These two residues are conserved in all plant ROPs but do not occur in any other Rho-type GTPases [11,17]. The phosphorylation takes place at several Ser/Thr residues, excluding S74, and the phospho-mimetic mutation of one of these residues affects the protein–protein interaction capability and the function of the GTPase These data open new avenues to investigate the interlink between kinase and ROP GTPase signalling pathways in plants
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