Characterization of ROP GTPase-activated Arabidopsis receptor-like cytoplasmic kinases (RLCK class VI_A)

Jurca Elena Manuela
Characterization of ROP GTPase-activated Arabidopsis receptor-like cytoplasmic kinases (RLCK class VI_A).
Doktori értekezés, Szegedi Tudományegyetem.

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Plants have to respond and adapt to a variety of continuously changing environmental factors in order to establish an appropriate developmental strategy to ensure survival. There are ample data showing that protein phosphorylation/dephosphorylation plays a central role in cellular signal transduction in all organisms (Herrmann et al. 2006; Stone and Walker 1995). Interestingly, plants have a similar system as mammals to detect and transfer signals across the cell membrane into the nucleus where adaptations could be initiated. For the detection and transfer of an external signal, mammalian systems have receptor protein kinases. The proteins contain a single hydrophobic transmembrane domain, an extracellular domain and protein kinase domain. The majority of animal receptor kinases are phosphorylated on tyrosine residues within the kinase domain (receptor tyrosin kinases or RTKs; Ullrich and Schlessinger 1990), but a few were discovered which are phosphorylated on serine and threonine residues (Lin et al. 1992). In plants, two different types of transmembrane receptor kinases are known, including receptor-like serine/threonine (Ser/Thr) kinases (receptor-like kinases RLKs; Shiu and Bleecker 2001, 2003; Shiu et al. 2004; Walker 1994), structurally similar to mammalian RTKs, and receptor histidine (His) kinases (Grefen and Harter 2004; Mizuno 2005; Urao et al. 2000). Since the first RLK-encoding gene family was found in Zea mays (Walker and Zhang 1990), thousands of RLK genes have been identified from many different plant species. The Arabidopsis genome contains more than 600 members, representing nearly 2.5% of the annotated protein-coding genes; and more than 1000 members were annotated in the rice genome (Shiu et al. 2004). Approximately 25% of the Arabidopsis RLKs contain only a kinase domain with no apparent signal sequence or transmembrane region and thus were collectively named as receptor like cytoplasmic kinases (RLCKs). Arabidopsis RLCKs can be subdivided into 12 classes with 193 protein coding genes all together. Concerning the function of plant RLCKs, at the present only few members have been characterized and it is very likely that they play major role in the perception and 93 transmission of external signals perceived by RLKs (Zhou et al. 1995; Murase et al. 2004). Recently, our group as well as a group in Germany reported a direct interaction of plant ROP GTPases with receptor-like cytoplasmic kinases (RLCK class VI) from Arabidopsis (Molendijk et al. 2008) and alfalfa (Dorjgotov et al. 2009). Moreover, we provide evidences that kinases belonging to the RLCK Class VI family of Medicago truncatula and Arabidopsis thaliana can be specifically activated by GTP-bound ROP GTPases in vitro further supporting the view that plant Rho (ROP) G-proteins may directly regulate downstream kinase signaling. A further kinase designated as cysteine-rich receptor kinase (NCRK) belonging to a distinct kinase family has also been shown to interact with ROPs (Molendijk et al. 2008). None of these plant specific ROP-interacting kinases has any characteristic domain or motif that could be correlated with their ability to bind ROP GTPases. Plant specific ROP GTPases are versatile molecular switches in many processes during plant growth, development and responses to the environment and thus a possible implication of RLCKs in these ROP-dependent signal transduction pathways is in discussion. Our general aim was to characterize the members of the Arabidopsis thaliana RLCK Class VI family of protein kinases. Despite of their potential significance in ROP GTPase mediated signaling, hardly any functional information was available until now about the fourteen Arabidopsis RLCK Class VI members. Sequence comparison and phylogenetic analysis revealed that gene duplication played a significant role in the formation of this kinase family and allowed the separation of the 14 RCLK VI kinases into two groups with seven members each (A1 to A7 and B1 to B7). The proteins are highly homologous to each other, especially in the kinase domain, but are divergent from the related kinase families. It was established that, several members have an N-terminal UspA (“universal stress protein”) domain (group B members) or an N-terminal serine-rich region (group A members). In order to formulate a possible biological role of AtRLCK_VI kinases, real-time quantitative reverse transcription-polymerase reaction (qRT-PCR) was used to determine relative transcript levels in the various organs (root, rosette leaves, cauline leaves, 94 inflorescence stem, flower buds, open flowers, siliques. exponentially dividing cultured cells) of the Arabidopsis plant as well as under a series of abiotic stress/hormone (osmotic, sugar, salt stress, oxidative stress, cold and hormone treatment) treatments in seedlings. AtRLCK VI genes exhibited diverse expression patterns in the various plant organs as well as in response to stress/hormone treatments. In certain cases, the paralogous genes retained a similar relative expression pattern. The presented data indicate that the activity of several members of the RLCK VI kinase subfamily is regulated at the transcriptional level during plant development as well as in response to environmental stresses. Their tightly regulated and generally low expression may indicate that these proteins are involved in specific rather than general cellular processes. As it was mentioned above, we showed that members of the Medicago and Arabidopsis RLCK VI family are specifically activated by ROP GTPases (Dorjgotov et al. 2009). In order to strengthen the view that RLCK VI members may indeed serve as ROP effectors in planta, the co-expression of ROPs and RLCK VI_A kinases was analysed in silico based on microarray data sets. Moreover, to have a full picture on potential partners involved in given ROP-dependent signaling pathways, all known ROP regulators and effectors were included into the study. The hierarchical clustering analysis of microarray expression data showed clear correlation of the relative expression pattern of several of the investigated proteins with organ/tissue specific expression. Five main expression clusters (A-E) could be established. These clusters include genes with preferential or more abundant expression in the pollen and flower; stem, node and hypocotyl; root; shoot and root tip; flower and silique; and all over the plant. In order to test RLCK-ROP interaction specificity a yeast two hybrid interaction matrix was established using ten RLCK VI kinases (5-5 from groups A and B, respectively) and eight Arabidopsis ROP GTPases. The interaction was found to be specific for the RLCK VI group A kinases only. However, in an in vitro pull down assay a randomly selected kinase from the group B showed also interaction with a ROP GTPase. This difference in the results obtained by the various protein-protein interaction approaches may be related to the differing conditions stabilizing or weakening the 95 interaction. However, based on the in vitro kinase activity assays (see further), we suppose that the interaction pattern obtained in the yeast assay is more specific and can better simulate the in planta conditions. Primary sequence analysis revealed several characteristic differences in the sequences of the kinases belonging to group A that showed interaction with ROPs in yeast two hybrid assays. In this way several residues/motives could be identified that may have a role in the AtRLCK-ROP binding. Inspite of the potential interactions of ROPs with various RLCK kinases in vitro, the kinase activity assays clearly indicated specificity towards a functional interaction between ROPs and only the A group of RLCK VI kinases. One kinase which belongs to the group B (AtRLCK VI_B3) and an other one from RLCK class VII, that were also tested, could not be activated by the AtROP1 GTPase in contrast to several RLCKVI_A members indicating ROP’s specificity towards RLCK VI_A kinases. The presence of the GTPases in the GTP-bound conformation is the most efficient in increasing the phosphorylation activity of the kinase. Based on the fact that RLCK VI_A kinases are preferentially and strongly activated by the GTPase in active conformation, it could be hypothesized that these kinases are potential downstream ROP GTPase effectors. This possibility was disapproved by Molendijk et al. (2008) first reporting ROP-RLCK interactions. They could not observe ROP GTPase-dependent in vitro autophosphorylation or AtROP4 substrate phosphorylation with ROP-interacting RLCK kinases immunoprecipitated from insect cells where they were co-expressed. Although it is likely that RLCK VI_A kinases serve as ROP effectors, it is not clear how the GTPases can activate the kinases. We know a bit more as the region of the ROP GTPase involved in kinase activation is considered. A specific region of RhoGTPases, the so called Rho-insert region, is implicated in effector binding and activation (Karnoub et al. 2004). Plant ROP GTPases have an insert region with characteristic differences as compared to human or yeast Rac, Rho and Cdc42 proteins that is an indication that plants have evolved specific ROP GTPase effectors (Berken, 2008). In our laboratory it was identified that this region is important for the activation of the MtRRK1 (MtRLCK VI_A2) kinase (Dorjgotov et al. 2009). 96 The biological role of functional ROP-RLCK interaction needs to be determined. In this direction we further proceed with the production of transgenic plants overexpressing or silencing the RLCK VI_A2 gene. Cell polarization is intimately linked to plant development, growth, and responses to the environment. It is well established that plant cells use conserved mechanisms such as Rho family GTPases (ROPs) to integrate both plant-specific and conserved polarity cues and to coordinate the cytoskeleton dynamics/reorganization and vesicular trafficking required for polarity establishment and maintenance. We suspect that AtRLCK VI_A2 as a potential ROP effector may have a role in polarity establishment. Directional cell expansion (e.g. trichomes) and tip growth (e.g. pollen tubes) are two basic processes underlying the morphogenesis of polar cell types in plants. Our preliminary data, that need further confirmation, indicated that both overexpressing as well as silencing of the gene disturb polar growth of the pollen tube and might affect trichome branching. Over-expression of AtRLCK VI_A2 increase the frequency of trichomes with fewer branches while silencing of the gene may lead to the apparition of trichomes with more branches, although only with a low frequency. Overexpressing AtRLCK VI_A2 in the pollen tube may result in the formation of bifurcated pollen tube tip. Silencing of AtRLCK VI_A2 could lead to the formation of ballooned pollen tube tips. Further more detailed investigations will be carried out to confirm the potential role of RLCK VI_A kinases in these and further ROP GTPase-regulated processes (e.g. pathogene response, ROS production) to support the view that RLCKs are indeed plant specific ROP GTPase effector kinases. In summary, we established that AtRLCK VI_A kinases interact and are activated in vitro by AtROP GTPases. Furthermore, the characterization of expression patterns and transgenic plants allowed us to hypothesize that these kinases may serve as ROP GTPase effectors in plants. This signaling step has several plant-specific aspects. Further studies will allow interesting insights into the evolution of cellular signaling mechanisms and will provide valuable new information about the regulation of plant development.

Mű típusa: Disszertáció (Doktori értekezés)
Doktori iskola: Biológia Doktori Iskola
Tudományterület / tudományág: természettudományok > biológiai tudományok
Magyar cím: A ROP GTP-ázok által aktivált lúdfű receptor-szerű citoplazmatikus kinázok (RLCK VI_A osztály) jellemzése
Idegen nyelvű cím: Characterization of ROP GTPase-activated Arabidopsis receptor-like cytoplasmic kinases (RLCK class VI_A)
Témavezető neveBeosztás, tudományos fokozat, intézményEmail
Dr. Fehér Attilatudományos tanácsadó, MTA doktora, MTA SZBK Növénybiológiai Inté
EPrint azonosító (ID): 870
Publikációban használt név : Jurca Elena Manuela
A mû MTMT azonosítója: 2774795
doi: 10.14232/phd.870
A feltöltés ideje: 2011. jún. 08. 14:23
Utolsó módosítás: 2017. jún. 13. 13:50
Védés állapota: védett

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