Role of Two Novel Pollen Tube Proteins in Polarized Rho GTPase Signalling

Lead Research Organisation: University of Warwick
Department Name: Warwick HRI


Polarization, a process that allows cells to organize structurally and functionally distinct poles, is essential not only for the normal behaviour and replication of single cells, but also for the development of multicellular organisms. The accumulation of active Rho GTPases at specific domains of the plasma membrane surrounding cells plays a key role in polarization. Rho GTPases are proteins that can regulate a variety of cellular process by controlling the activity of other proteins with which they directly interact (effectors). The molecular mechanisms that control the local accumulation of active Rho GTPases at specific membrane domains are not well understood, and how these proteins regulate cellular processes has only been clarified to a limited extent. Tobacco pollen tubes are an exceptionally useful experimental model system to investigate polarization. A Rho GTPase called Nt-Rac5 specifically accumulates at the membrane at one end of these highly elongated cells, where it controls cell expansion that occurs at this location. We have identified two novel tobacco pollen tube proteins, whose activities appear to depend on stimulation by Nt-Rac5, as suggested by the results of preliminary experiments. These experiments have shown that Nt-Rak1 directly binds to active Nt-Rac5, and is therefore likely to act as an effector of this protein. Nt-Rak1 has the ability to regulate the activity of other proteins by transferring phosphate groups to them, a process called phosphorylation. Interestingly, we have found that Nt-Rak1 not only interacts with Nt-PubPT, a putative target protein we have identified, but also phosphorylates Nt-Rac5. This suggests that Nt-Rak1 acts as a Nt-Rac5 effector, which regulates the function of Nt-PubPT, and at the same time modulates the activity of Nt-Rac5. The modulation of the activity of a protein by its own effector is called feedback regulation, a process that is often involved in polarization. With this proposal, we ask for funding to further investigate and establish the functions of Nt-Rak1 and Nt-PubPT in the regulation of the activity of Nt-Rac5 at the expanding end of tobacco pollen tubes, and in the control of cellular processes by this protein. We believe that this work will enhance out understanding of the molecular mechanisms underlying polarization, a process with essential functions in single cells and multicellular organisms.

Technical Summary

To enhance our understanding of the spatial control of Rho GTPase activity and cellular polarization in eukaryotes, as well as of Rho dependent signalling in plants, we propose to functionally characterize two novel tobacco pollen tube proteins: Nt-Rak1 and Nt-PubPT. This project aims to build on preliminary data and to establish that the protein kinase Nt-Rak1 acts as an effector of the tobacco pollen tube Rho GTPase Nt-Rac5, controls the E3 ubiquitin ligase Nt-PubPT, and feed-back regulates Rho GTPase activity. We propose to test biochemically whether Nt-Rak1, which specifically interacts with activated Nt-Rac5 in yeast two hybrid assays and in vitro, feedback regulates Nt-Rac5 by phosphorylating either directly this protein, or regulatory factors that control Nt-Rac5 activity. Biochemical experiments will also be performed to demonstrate that Nt-PubPT, which interacts with Nt-Rak1 in yeast two-hybrid assays, binds to this protein in vitro, and is a target of its kinase activity. To establish Nt-Rak1 and Nt-PubPT functions in Nt-Rac5 signalling in vivo, we plan to employ cell biological approaches. Based on fluorescent protein tagging and confocal imaging, we will determine the localization of Nt-Rak1 and Nt-PubPT in pollen tubes during normal growth and after the inactivation of Nt-Rac5 signalling. Colocalization of these proteins with each other and with Nt-Rac5 will also be studied. A quantitative comparison of effects of transient overexpression of Nt-Rac5, Nt-Rak1 or Nt-PubPT in tobacco pollen tubes, either alone or in combination with other factors involved in Rho signalling, will complement these experiments. We expect strong support for the results of the experiments described above to result from analysing effects of RNAi mediated knock-down of Nt-Rak1 and Nt-PubPT expression in tobacco pollen tubes. The investigation of effects of overexpessing Nt-Rak1 or Nt-PubPT in Nt-Rac5 or Nt-Rak1 RNAi pollen tubes will also be highly informative.


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Description The Rho family of small GTPases plays a key role in controlling the directional expansion of pollen tube cells, in an actin-dependent and tip-directed secretion process. Preliminary work suggested that a receptor-like kinase Nt-Rak1 is specifically expressed in tobacco pollen tubes, and plays a pivotal role in regulation of the activated GTPase Nt-Rac5 in pollen tube growth of tobacco. Yeast two-hybrid screens using Nt-Rak1 as bait had also identified targets of Nt-Rak1 kinase activity, including a putative E3 ubiquitin ligase Nt-PubPT. Thus, our objectives were to characterize functions of Nt-Rak1 in the feedback regulation of Nt-Rac5, and to analyse the role of Nt-PubPT as a target of Nt-Rak1 in Nt-Rac5 downstream signaling.

Our data (described below) indicates that Nt-Rak1 is recruited to the apical pollen tube plasma membrane specifically by activated Nt-Rac5. Nt-Rak1 phosphorylates itself, Nt-Rac5 and downstream signaling targets including Nt-PubPT. The main target site of Nt-Rak1 mediated phosphorylation of Nt-Rac5 is S193, which lies within the protein-binding region (PBR). Phosphorylation of this residue has no effect on Nt-Rac5 regulation by two proteins (Nt-RopGAP1 or Nt-RhoGDI2) but disrupts surface charge interactions required for Nt-Rac5 membrane association. Thus, Nt-Rak1 appears to function as Nt-Rac5 effector, which at the same time attenuates Rac5 signaling via negative feedback regulation in pollen tube growth.

For co-localization, fluorescent Nt-Rac5 and Nt-Rak1 fusion proteins were observed in living tobacco pollen tubes and with bimolecular fluorescence complementation. We used phosphor-peptide mapping to identify three N-Rac5 residues (S97, T139 and S193), which are phosphorylated by Nt-Rak1. Replacement of phosphorylated S97 or T139 residues (outside of domains involved in Nt-Rac5 function) by alanine resulted in a 20-30% reduction of Nt-Rak1 mediated Nt-Rac5 phosphorylation; whereas replacement of S193 (located within the C-terminal polybasic region) by alanine caused an 80% reduction of phosphorylation. Rho GTPases require GTPase activating proteins (GAPs) to promote GTP hydrolysis. Experiments showed that neither intrinsic nor Nt-RhoGAP1-stimulated GTPase activity of Nt-Rac5 was affected when the S193 site was replaced by alanine (S193A); or by either aspartic acid (S193D) or glutamic acid (S193E) to mimic phosphorylation. Guanine nucleotide dissociation inhibitors (GDIs) are negative regulators of Rho signaling. GDIs contain hydrophobic pockets capable of accommodating the prenyl tail of Rho GTPases, and can transfer Rho GTPases from the plasma membrane to the cytoplasm. A pull-down assay showed that the interaction of prenylated Nt-Rac5 with the tobacco pollen tube GDI Nt-RhoGDI2 was not affected when the main target site of Nt-Rak1 kinase activity was mutated to abolish (S193A) or mimic (S193D or S193E) phosphorylation. Phosphorylation by Nt-Rak1 had no effect on the in vitro interaction of prenylated Nt-Rac5 with Nt-RhoGDI2. Function of these proteins depends on interactions between the negatively charged cytoplasmic surface of the plasma membrane and the positively charged PBR adjacent to the C-terminal CAAX domain of Rho GTPases. GFP tagging showed that mutating Nt-Rac5 to abolish phosphorylation of the main target site of Nt-Rak1 kinase activity (S193A; positioned within the PBR) strongly enhanced Nt-Rac5 association of with the apical plasma membrane of pollen tubes. Mutating the same site to mimic phosphorylation (S193D or S193E) abolished Nt-Rac5 membrane association. The C-terminal fragment of Nt-PubPT identified by yeast two-hybrid screen was used to clone a full length cDNA of Nt-PubPT. This was difficult and not achieved until late in the final year. However, in vitro phosphorylation assays showed that full-length Nt-PubPT is phosphorylated by Nt-Rak1, confirming that Nt-PubPT is likely to act downstream of Nt-Rak1 in an Nt-Rac5 dependent signaling pathway.
Exploitation Route The lead PI moved back to Germany, and has been using the materials and results to continue his research on the topic.
Sectors Agriculture, Food and Drink