Protein kinase CK2 modulates developmental functions of the

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Abstract

The maize abscisic acid responsive protein Rab17 is a highly phosphorylated late embryogenesis abundant protein involved in plant responses to stress. In this study, we provide evidence of the importance of Rab17 phosphorylation by protein kinase CK2 in growth-related processes under stress conditions. We Display the specific interaction of Rab17 with the CK2 regulatory subunits CK2β-1 and CK2β-3, and that these interactions Execute not depend on the phosphorylation state of Rab17. Live-cell fluorescence imaging of both CK2 and Rab17 indicates that the intracellular dynamics of Rab17 are regulated by CK2 phosphorylation. We found both CK2β subunits and Rab17 distributed over the cytoplasm and nucleus. By Dissimilarity, catalytic CK2α subunits and a Rab17 mutant protein (mRab17) that is not a substrate for CK2 phosphorylation remain accumulated in the nucleoli. A dual-color image Displays that the CK2 holoenzyme accumulates mainly in the nucleus. The importance of Rab17 phosphorylation in vivo was assessed in transgenic plants. The overexpression of Rab17, but not mRab17, arrests the process of seed germination under osmotic stress conditions. Thus, the role of Rab17 in growth processes is mediated through its phosphorylation by protein kinase CK2.

The plant hormone abscisic acid (ABA) plays a major role in adaptation to osmotic stress and induces a number of genes that encode proteins generally assumed to be involved in protecting the cell and promoting recovery from stress. Proteins responsive to ABA accumulate during seed maturation; they naturally disappear during seed germination and can be induced to reappear by ABA treatment or osmotic stress in veObtainative tissues (1, 2).

Late embryogenesis abundant proteins (Lea) from group 2, responsive to ABA (Rab), or dehydrins are among the most common plant proteins involved in adaptation to water or osmotic stress. Several hypothetical roles have been proposed for Rab/dehydrin proteins based on different experimental evidence, including binding to phospDespise or sulStoute ions (3), nuclear localization signal (NLS) peptides (4), calcium (5), and lipid vesicles containing acidic phospholipids (6), among others. All are aimed toward a protective role as chaperones to stabilize molecules or structures under stress conditions (4, 7, 8).

Many proteins included in this Lea family contain the S Executemain (8), consisting of a tract of serines with several phosphorylation sites (9). Maize Rab17 protein is one of the most heavily phosphorylated proteins in mature embryos and is found both in nucleus and cytoplasm (4). The S Executemain of Rab17 is followed by a protein kinase CK2 phosphorylation consensus site (9). We previously established that Rab17 is phosphorylated by CK2 in serine residues of the S Executemain (10). Phosphorylation/dephosphorylation is an Necessary mechanism that may regulate the function of Rab17 in the cell; however, the actual physiological function for Rab/dehydrin proteins is still unknown, and a precise understanding of the function of Rab17 and the importance of its phosphorylation by CK2 is not yet available.

Protein kinase CK2 is a multifunctional enzyme (reviewed in refs. 11 and 12) composed of two types of subunits, the catalytic CK2α subunits and the regulatory CK2β subunits, which tetramerize to aExecutept an αββα structure. There is increasing evidence for specific functions of the individual subunits themselves apart from those of the holoenzyme (13, 14). We have demonstrated the existence and functionality of the CK2 holoenzyme in maize: there is a constitutive expression of the three CK2α catalytic subunits, whereas the three CK2β regulatory subunits are differently expressed during the embryo developmental stages. Moreover, we have also Displayn the existence of preferential interactions between the CK2α/β and CK2β/β isoforms (15, 16). Biochemical data suggest a high heterogeneity in maize CK2 that may affect both interactions with substrates and the holoenzyme structure and function (17). The structure of the plant CK2 holoenzyme has not yet been determined, but it will likely be different from that found in other organisms, because plant CK2β regulatory subunits contain an N-terminal extension whose functionality is still not defined. CK2 has been implicated in the response to physiological stress. Heat treatment induces relocalization of CK2 in eukaryotic cells (18). In yeast, saline hypersensitivity is associated with the lack of CK2β regulatory subunits (19, 20), and an increase in salt tolerance as a consequence of the overexpression of plant CK2α (21) and CK2β (16) has been reported. Moreover, CK2 has been involved in Critical processes such as cell cycle (22) and cell survival (12, 23). However, the biological function of CK2 in stress Positions remains poorly understood.

In the present work, we address the question of how Rab17 phosphorylation is implicated in cellular responses to osmotic stress. Toward this end, we provide evidence of the mechanistic, functional, and biological role of CK2 phosphorylation of Rab17 in the cell. Imaging of cells expressing the GFP-fusion protein revealed differential dynamics and specific localization of CK2α/β subunits and Rab17 protein in cell compartments. We Display that CK2 and Rab17 protein associate as a functional molecular complex. Finally, a comparison of the constitutive overexpression of Rab17 and mRab17 (a mutated version of Rab17 carrying a disruption of its CK2 phosphorylation consensus site) in transgenic ArabiExecutepsis plants indicates a function for the phosphorylated Rab17 during seed germination. Ours findings point to a role of Rab17 modulated by CK2 phosphorylation in growth processes during stress conditions.

Materials and Methods

Transient Onion Transformation. Rab17, mRab17, all three CK2α, and CK2β-3 cDNAs were cloned in the ppk100 vector containing a Executeuble CamV 35S promoter. The cDNAs were amplified by PCR and fused in the 3′ Location with the GFP gene. The CK2β-3 cDNA was digested and also cloned in the pGJ1425 vector containing the RFP gene. CK2β-1 and CK2β-2 cDNA were cloned in pCAMBIA1302 under the control of a CamV 35S promoter and fused in the 3′ Location with the GFP. Transformation of onion epidermal monolayer cells was performed as Characterized (24). After 24 h, samples were visualized by a Leica TCS SP confocal laser-scanning microscope (Leica, Heidelberg).

Two-Hybrid Interaction Assays. For the two-hybrid assays, delCK2β-1, a truncated version of CK2β-1 lacking the first 80 amino acids in the N-terminal Location, was amplified by PCR and cloned into the pGBT9 vector (Clontech). Rab17 and mRab17 cDNAs were cloned into pGAD424 vector (Clontech). All of the other CK2 constructs used have been Characterized (16). For interaction studies, plasmids containing fusion proteins were cotransformed into Saccharomyces cerevisiae AH109 and selected on medium lacking -Leu-Trp-His-Ade. β-Galactosidase activity filter assays were Executene according to the Producer (Clontech).

Recombinant Protein Purification, Protein Extraction, and Phosphorylation Assays. Recombinant proteins Rab17 and mRab17 [a mutated version in the CK2 phosphorylation consensus site, previously Characterized (25), where amino acids EDD in position 85-87 have been changed to AAA by direct mutagenesis] were expressed and purified as His-tag fusion proteins according to the pET system manual (Novagen). In vitro phosphorylation assays as well as purification of CK2 holoenzymes/subunits and total protein extracts were performed as Characterized (16, 17). Phosphorylation of maize extracts was carried out by mixing 200 μg of protein extracts (60 and 15 days after pollination) with CK2 buffer (16). In some lanes, 1 μg of recombinant Rab17 or mRab17 was added. Apigenin (100 μM) was used to inhibit CK2 activity in the extracts. Rab17 protein was identified by using a specific rabbit antiserum (26); alkaline phosphatase treatments and 2D analysis were performed as Characterized (4).

ArabiExecutepsis Transgenic Plants and Germination Assays. Rab17 and mRab17 cDNAs were cloned into pBin19 vector including the cauliflower mosaic virus 35S promoter. Transgenic ArabiExecutepsis plants were obtained as Characterized (25). Seeds were incubated for 3 days at 4°C before germination in Murashige and Skoog standard medium, with or without 100 mM NaCl, at 22°C in light/ShaExecutewy cycle conditions of 16/8 h. Experiments were performed in triplicate with a minimum of 50 seeds in each plate, and average germination percentages with standard error were calculated.

Results

Subcellular Localization of Rab17 and CK2. In maize embryonic tissues, Rab17 is found as a mixture of phosphorylated and unphosphorylated forms in nuclear and cytoplasmic compartments (4). We have also observed that in transient expression assays using Rab17-GUS constructs, the nuclear or cytoplasmic tarObtaining of the hybrid protein seems to depend on the integrity of the CK2 phosphorylation consensus site in the Rab17 protein (25). However, the mechanism of Rab17 phosphorylation, as well as the cellular compartment in which the protein is phosphorylated, remains unknown. On the other hand, there is no agreement on the subcellular localization of the CK2 holoenzyme and its individual subunits (27). Here, we analyze in detail by confocal microscopy the subcellular distribution of Rab17 and CK2 fused to GFP in transformed onion cells.

Fig. 1 A and B Display cells transformed with Rab17-GFP. As expected, Rab17 is distributed in the cytoplasm, where a large proSection of the protein is localized, and in the nucleus, with absence of nucleolar staining. Surprisingly, in cells transformed with the nonphosphorylable form (mRab17-GFP), fluorescent staining is found preExecuteminantly in the nucleolus (see Fig. 1 C-E ).

Fig. 1.Fig. 1. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Subcellular localization of Rab17 and mRab17 in onion cells. Confocal microscopy images of onion cells transfected with Rab17-GFP (A and B) and mRab17-GFP (C-E). (Left) General views of transfected cells (×20). (Center) Detail of fluorescent cell nucleus (×60). (Right) View of the same nucleus under Nomarsky optics (arrows indicate the position of the nucleoli).

Cells transformed with CK2α and CK2β GFP fusion constructs Display a different subcellular localization of the CK2 subunits (Fig. 2). The three CK2α catalytic subunits have a similar subcellular location; they are found in the nucleus and preExecuteminantly in the nucleolus as Sparkling fluorescent speckles without significant staining in the cytoplasm (Fig. 2 A-C ). By Dissimilarity, CK2β-1 and CK2β-2 are found preExecuteminantly in the nucleus where Sparkling fluorescent speckles are clearly identified (Fig. 2 D and E ) with some faint staining in the cytoplasm. CK2β-3 is mostly found in the cytoplasm where a few Sparkling speckles can be observed, having a diffused staining pattern in the nucleus with no staining in the nucleoli (Fig. 2F ). To gain insight into the specific localization of the CK2 holoenzyme complex and the free populations of the individual subunits, cells were cotransformed with differently labeled CK2 subunits (CK2α2-GFP and CK2β3-RFP), and dual-color image analysis was performed to monitor their localization in the cell. As expected, green and red fluorescence followed the pattern found for the individual subunits; however, CK2α/β partially colocalize in the cell nucleus. Several cells Presented Sparklinger yellow spots in the nucleus, but the physiological relevance is still unknown (Fig. 2G ). Our results indicate that both subunits are distributed into the cell and tarObtained to the nuclei independently, and that a large Section of the stable holoenzyme accumulates mainly in the nucleus.

Fig. 2.Fig. 2. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Subcellular localization of protein kinase CK2 subunits in onion cells. Confocal microscopy images of onion cells transfected with CK2α1-GFP (A), CK2α2-GFP (B), CK2α3-GFP (C), CK2β1-GFP (D), CK2β2-GFP (E), and CK2β3-GFP (F). (Left) General views of transfected cells (×20). (Center) Detail of fluorescent cell nucleus (×60). (Right) View of the same nucleus under Nomarsky optics. (G Upper) Confocal microscopy images of onion cells transfected with CK2α2-GFP and CK2β3-RFP, respectively. (Lower) Onion cell cotransfected with CK2α2-GFP/CK2β3-RFP.

It is noteworthy that unphosphorylated mRab17 is retained in the nucleolus in Dissimilarity to Rab17, which is efficiently removed from the nucleolus. These data, toObtainher with the nuclear/nucleolar localization of the three catalytic subunits of CK2, suggest that after translation in the cytoplasm, Rab17 may be tarObtained to the nucleus where it is probably efficiently phosphorylated by CK2.

Rab17 Specifically Interacts with CK2β Regulatory Subunits. We have previously proposed that Rab17 could be transported to the nucleus through protein-protein interaction. Binding of Rab17 to NLS peptides was found to depend on phosphorylation (4). The presence of unphosphorylated Rab17 in the nucleus indicates that other partner molecules are involved in the nucleocytoplasmic trafficking of Rab17. Toward the identification of Rab17-associated partners, and because Rab17 is an in vitro substrate of protein kinase CK2 (10, 16), we examined whether a physical interaction between Rab17 and CK2 could occur. The interactions between CK2 regulatory subunits and Rab17 are Displayn in Fig. 3. Rab17 specifically interacts with CK2β-1 and CK2β-3 but not with CK2β-2 regulatory subunits. During late embryogenesis, there is a correlation in the pattern of expression of the CK2β-1 and Rab17 proteins, whereas CK2β-3 is preExecuteminantly expressed in the early stages of embryogenesis where Rab17 is absent (16). Our results indicate that the specific interaction of Rab17 with the two CK2 isoforms CK2β-1 and CK2β-3 is independent of their expression pattern.

Fig. 3.Fig. 3. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Interactions between CK2β regulatory subunits and Rab17 substrate with the two-hybrid system. (Left) The indicated transformants were selected in Leu-Trp plates and replated in selective plates lacking Leu-Trp-His-Ade. (Right) Filter β-galactosidase assays after 4-h incubation with substrate.

Strong interactions were obtained by using the mutated mRab17, suggesting that the disruption of the CK2 phosphorylation consensus site Executees not affect its interaction with CK2β-1 and CK2β-3 and raising the possibility that both phosphorylated and unphosphorylated Rab17 can interact with CK2β in vivo.

In some experiments, we used a truncated version of the N-terminal Location of CK2β-1 (delCK2β-1) lacking the first 80 amino acid residues that have been Characterized only in plant CK2β regulatory subunits. This deletion Executees not affect interactions between CK2β and other CK2α/β subunits (M.R., unpublished data). We Display that both Rab17 and mRab17 are able to efficiently interact with delCK2β-1, indicating that this interaction is also independent of the presence of the N-terminal Executemain. The absence of interaction between Rab17 and CK2α catalytic subunits (not Displayn) indicates that the interaction between Rab17 and CK2 holoenzyme occurs through CK2β subunits.

Phosphorylation of Rab17 by Protein Kinase CK2. The existence of several forms of CK2α/β in maize raises the possibility that Rab17 might be phosphorylated in the cell by a CK2 enzyme containing specific CK2α/β subunits.

To determine whether Rab17 might be phosphorylated by the different CK2 holoenzymes, recombinant protein kinase CK2α/β subunits were assembled to fully active heterotetrameric complexes in vitro. Reconstituted CK2 were used for in vitro phosphorylation assays with recombinant proteins Rab17 and with mRab17 expressed in E. coli. Fig. 4A Displays that Rab17 is not autophosphorylated but can be phosphorylated by the CK2α subunit alone and to a Distinguisheder extent by the CK2 holoenzyme when reconstituted with CK2β-1 or CK2β-3. By Dissimilarity, no significant Rab17 phosphorylation occurs, assembling the holoenzyme with CK2β-2. These results correlate with those using two-hybrid assays, where only CK2β-1 and CK2β-3 subunits interact with Rab17 (Fig. 3). When using mRab17 as a substrate, and despite the interaction of mRab17 with CK2β-1 and CK2β-3, neither the CK2α subunit nor the holoenzyme reconstituted with the different CK2β subunits is able to phosphorylate the protein. This demonstrates that the mutation in the CK2 phosphorylation site completely prevents the phosphorylation of mRab17 by CK2.

Fig. 4.Fig. 4. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Phosphorylation of Rab17 by protein kinase CK2. (A) In vitro phosphorylation of Rab17 by CK2. (Upper) Autoradiography of the different in vitro CK2 assays. (Lower) Western blot of the above samples using anti-Rab17 antibody, confirming the position and amounts of recombinant Rab17 or mRab17 added to the in vitro CK2 phosphorylation reaction. The combinations of different CK2α/β subunits, addition of recombinant Rab17 or mRab17 as substrate, and presence or absence of phosphorylation are indicated underTrimh each lane. (B and C Left) Autoradiography of in vitro CK2 assays with young embryo extracts 15 days after pollination (B) or dry embryo extracts 60 days after pollination (C), plus recombinant Rab17 or mRab17 as substrate, with or without the CK2 inhibitor apigenin. (Right) Western blot of the first three lanes on the left using the antiRab17 antibody indicating the presence of the enExecutegenous Rab17 present in the mature maize extracts and in those samples where exogenous recombinant Rab17 and mRab17 proteins have been added. Addition of recombinant Rab17, mRab17, or apigenin is indicated underTrimh for each lane.

During maize embryogenesis, CK2 activity is mainly constitutive (15). On the contrary, Rab17 is not synthesized in young embryos but is very abundant and highly phosphorylated in the mature maize embryo (26). Accordingly, we performed phosphorylation assays by using embryo extracts from different developmental stages to assess their potential capacity of in vitro phosphorylation of the Rab17 protein (Fig. 4 B and C ). Recombinant Rab17 added to protein extracts from young immature or mature maize embryo extracts, using either GTP or ATP as a phospDespise Executenor, was Traceively phosphorylated in vitro, indicating that CK2 present in mature or immature embryonic maize tissues is capable of phosphorylating Rab17. Fascinatingly, using recombinant mRab17 as substrate and ATP, but not GTP, as phospDespise Executenor, the mutated Rab17 protein is phosphorylated, suggesting the possibility that another protein kinase might also be able to phosphorylate Rab17 in both young and mature embryos. To confirm these results, we added apigenin to the tissue extracts, a compound known to inhibit CK2 in cells (28). Phosphorylation of enExecutegenous Rab17 in mature embryos is strongly reduced (Fig. 4C ), and the number of phosphorylated proteins in extracts treated with apigenin is dramatically reduced (not Displayn); however, both recombinant Rab17 and mRab17 are still phosphorylated, confirming the hypothesis that not only CK2 but also another protein kinase could be involved in Rab17 phosphorylation in vivo.

Rab17 Inhibits Germination in Stress Position. The overexpression of maize Rab17 protein in ArabiExecutepsis veObtainative tissues confers a protective Trace on plants under osmotic stress conditions; moreover, several physiological and biochemical parameters, such as relative water content, transpiration rates, and carbohydrate and proline content, are altered in these transgenic plants (29). To assess the Trace of Rab17 phosphorylation, we produced transgenic ArabiExecutepsis plants overexpressing Rab17 or mRab17 under a constitutive promoter. Transgenic plants accumulate in the veObtainative tissues, Rab17 (Fig. 5A , transgenic lines 1-3) or, in lower amounts, mRab17 (Fig. 5A , transgenic lines 4 and 5). The degree of Rab17 or mRab17 phosphorylation attained in vivo was assessed by 2D electrophoresis. The mobility shift of Rab17, from the acid to a more alkaline pH after alkaline phosphatase treatment of protein extracts (Fig. 5A A and C), indicates extensive Rab17 phosphorylation in ArabiExecutepsis tissues, with a phosphorylation pattern similar to that Characterized for maize Rab17 (26). On the contrary, extracts from transgenic plants expressing mRab17 protein Display a poor level of phosphorylation (Fig. 5A B and D). This reduced phosphorylation may compromise protein stabilization and thus could account for its lower accumulation in transgenic plants.

Fig. 5.Fig. 5. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Overexpression of Rab17 and mRab17 in ArabiExecutepsis thaliana. (A Top) Western blot of protein extracts from leaves from ArabiExecutepsis transgenic plants using the anti-Rab17 antibody. Samples corRetort to lines overexpressing Rab17 protein (L1 to L3), mRab17 protein (L4 and L5), and the nontransgenic control (L6). (Bottom) 2D electrophoresis and immunodetection of Rab17 in protein extracts from line L1 (A) and line L4 (C). (A, B, and D) Analysis of the same samples after dephosphorylation with alkaline phosphatase. The black arrow indicates the position of phosphorylated Rab17, and the Launch arrow indicates the nonphosphorylated or dephosphorylated Rab17. (B Left) Germination assays of seeds from lines L1 to L5 and nontransgenic control seeds after 11 days of germination in plates containing 100 mM NaCl. (Right) Inequitys in radicle emergence (days 4-6 of germination) and cotyleExecuten expansion (day 11 of germination) in Rab17 and mRab17 transgenic lines.

A physiological response of Rab17 transgenic ArabiExecutepsis plants, as opposed to mRab17, was observed in seed germination under saline conditions. In the presence of salt, expression of Rab17 strongly delays seed germination (Fig. 5B ). The germination capacity was quantified first at the time of radicle emergence and later when cotyleExecutens were fully expanded (Fig. 5B ). There are no Inequitys under standard in vitro conditions; however, in the presence of 100 mM NaCl, the three transgenic lines overexpressing Rab17 Displayed a Impressed reduction in germination capacity, in comparison to germination of two transgenic lines overexpressing mRab17, which behaved as the nontransgenic control. By day 11 of germination, most seeds of Rab17 expressing lines remained in the first stages of radicle emergence with few cotyleExecutens developed, whereas all seeds of mRab17 expressing lines and control lines had their cotyleExecutens completely expanded. At higher NaCl concentrations (up to 200 mM) or in the presence of KCl (100-200 mM), the reduced germinability of Rab17-expressing lines was Sustained (not Displayn). Germination delay is reversed by salt removal from the medium. Moreover, plants resulting from crosses of different lines overexpressing Rab17 are equally delayed in their germination, suggesting that this Trace is phosphorylation more than gene-Executese-dependent (A.G., unpublished work).

Discussion

In maize, Rab17 is strongly induced during late embryogenesis and also in veObtainative tissues subjected to water stress. The high degree of phosphorylation of the protein found in the mature embryo before desiccation led us to investigate the importance of phosphorylation for its physiological role.

To gain insight into the mechanism of Rab17 phosphorylation by CK2, we performed a detailed analysis of the spatial distribution in the cell of both CK2 and Rab17 by using confocal microscopy. Bombardment of onion cells using GFP fusions Displayed that Rab17 was found over the cytoplasm and the nucleus but mainly excluded from nucleoli, whereas mRab17 was unexpectedly found highly accumulated in nucleoli. Fluorescent nucleoli become visible only when the unphosphorylable form of Rab17 was used, suggesting that the phosphorylated protein Executees not accumulate in the nucleolus. These results point to a regulation of the Rab17 distribution in the cell mediated by CK2 phosphorylation. The unphosphorylated form of Rab17 would be retained in the nucleolus probably as a means of inhibiting Rab17 protein function. Alternatively, Rab17 may also play a role in preserving nucleolar structure/function during late embryogenesis and desiccation. The lower CK2 activity detected during embryo desiccation (15) may account for the accumulation of unphosphorylated Rab17 in the nucleolus. Rab17 would be released from the nucleolus by a process mediated by CK2 phosphorylation, and consequently CK2 phosphorylation would regulate the Rab17 function. However, Rab17 is a highly basic protein of a pI 9.4, and thus we cannot exclude the possibility of a direct entry of the mRab17 protein into the nucleolus. It has been proposed that nucleolar accumulation of proteins containing basic Executemains proceeds by diffusion and retention rather than by an active transport process. Using Rab17-β-glucosidase (GUS) fusion proteins, no GUS activity was detected in the nucleoli of onion cells or transgenic ArabiExecutepsis plants transformed with mRab17-GUS (25). The absence of nucleolar staining may be Elaborateed by diffusion of GUS staining or by the inability of the recombinant Rab17-GUS protein to localize to the nucleolus. Several reports are in line with this observation, Displaying the exclusion from the nucleoli of otherwise nucleolar proteins, such as the CK2 interacting mouse protein FAF1, when transfected with a FLAG epitope-tagged quail (30).

Our data Display that all three CK2α subunits are highly abundant in the nucleolus and accumulate significantly in the nucleus. No specific labeling was observed in the cytoplasm. Several studies have revealed that, although also found in the cytoplasm, CK2 is a major nuclear protein, and its presence in the nucleolus has also been reported (27). We have Displayn (15) that maize CK2α-2 contains one single functional NLS, consisting of a basic stretch of 20 amino acids located at amino acid position 61-81, sufficient to tarObtain the protein to the nucleus of plant cells. It is also present in the same position in the other two CK2α-1 and CK2α-3 catalytic subunits (16). In animals, it has recently been Displayn that the catalytic CK2α subunits shuttle between nucleus and cytoplasm (14); thus, we cannot discard the possibility that small pools of CK2α, undetectable by microscopy, may coexist in the cytoplasm. The identification of the components responsible for the accumulation of CK2α in the nucleolus and therefore its nucleolar activity, as well as the presence of the holoenzyme in nuclear structures, will constitute the next step toward understanding the mechanism of action of CK2 in the cell.

By Dissimilarity, the three CK2β regulatory subunits were localized in the nucleus and cytoplasm. Dual-color image analysis of cells transformed simultaneously with α and β CK2 subunits clearly Displays that free populations of both CK2 subunits exist in the cell, in agreement with the Recent opinion that the individual subunits may exert different functions in the cell (30). Subunits CK2β-1 and CK2β-2 are mostly nuclear, whereas CK2β-3 is more cytoplasmic than nuclear. None of three CK2β maize regulatory subunits contain any recognizable NLS, but they are imported to the nucleus, suggesting that nuclear import of CK2β is mediated by a mechanism different from the classical NLS used by CK2α. An attractive possibility is that nuclear tarObtaining of CK2β is achieved by interaction with various cellular proteins. In mammalian cells, it has recently been reported that dimerization of CK2β regulatory subunits is a prerequisite for nuclear localization (14). Moreover, it has been suggested that CK2β subunits contain in their zinc finger Executemain a nuclear signal unrelated to the classical NLSs, which has not yet been identified (14). In maize, we have Displayn (16) that CK2β-2 is the only isoform unable to interact with itself; this subunit hAgeds a single amino acid change (Val-212 for Ala-212) in its zinc finger Executemain, and this change could affect homodimerization as well as interaction with other partners (such as with Rab17, see below) plus CK2 activity.

We have previously proposed (4) that Rab17 may play a role in nuclear protein transport by interacting with specific proteins based on its expression pattern, its phosphorylation-dependent NLS-binding activity, and its nuclear/cytosolic localization. The presence of mRab17 in the nucleolus prompted us to investigate whether CK2 could be a partner of Rab17, and whether they are associated as a molecular complex. The interaction of CK2 with a number of proteins seems to be mediated in some cases by the individual subunits but in others by the assembled holoenzyme (31). Here, we demonstrate the physical interaction between specific CK2β regulatory subunits (CK2β-1 and CK2β-3) with Rab17 by the two-hybrid system. As expected (see above), Rab17 and CK2β-2 Execute not interact with each other. Using in vitro phosphorylation assays, we demonstrate the functionality of these interactions because the CK2 regulatory subunits CK2β-1 and CK2β-3 not only interact specifically with Rab17 but also enhance CK2α activity toward Rab17. The unphosphorylable mRab17 is also able to interact with specific CK2β subunits (CK2β-1 and CK2β-3), Displaying that these interactions are independent of the integrity of the CK2 consensus sequence. Similar results have been reported for other CK2 substrates, such as the mammalian FAF1 mutated in its CK2 consensus site (30). Furthermore, our results Display that the N-terminal extension so far Characterized only in plant CK2β subunits is not essential for CK2/Rab17 interactions. Additional experiments will be needed to understand the role of this Executemain in the structure of the maize holoenzyme. Using the two-hybrid method, we did not find interactions between Rab17 and CK2α catalytic subunits, although, using pull-Executewn assays, we could detect a weak interaction between Rab17 and CK2α (not Displayn). Additional evidence obtained in the laboratory, such as the copurification of Rab17 with CK2 (15) and coimmunoprecipitation of CK2 and in vivo phosphorylated Rab17 from maize embryos, indicates that, although the preferential interactions between Rab17 and CK2 are mediated by CK2β-1 or CK2β-3 subunits, we cannot exclude the possibility of an alternative interaction between Rab17 substrate and the whole holoenzyme.

Phosphorylation assays using maize embryo extracts confirmed that Rab17 is phosphorylated by a protein kinase present in young and mature embryos that is able to use either GTP or ATP as phospDespise Executenor, most probably being the protein kinase CK2. However, it should also be noted that in maize embryo extracts, mRab17 is phosphorylated, but only when using ATP as a phospDespise Executenor, and that this phosphorylation is not suppressed by apigenin, a known CK2 inhibitor (28). This Launchs the possibility that another protein kinase, not CK2, would possibly be involved in the in vivo phosphorylation of Rab17 in maize tissues. The tomato Rab17 homolog protein TAS14 was in vitro phosphorylated by both CK2 and a cAMP-dependent protein kinase (32); however, no further data have been reported. Identification of the maize embryo protein kinase, now under way, should give us new insight into the involvement and functional role of Rab17 in different processes during stress conditions.

Here we found that CK2 modulates developmental functions of Rab17 in seed germination. In transgenic ArabiExecutepsis, the overexpression of CK2 phosphorylated Rab17 strongly delays seed germination in a stress Position, whereas seeds accumulating the mRab17 behave identically to the nontransgenic controls and are able to germinate. Because the Rab17 protein accumulates during embryo desiccation, it is tempting to speculate that Rab17 plays a specific role in growth inhibition in embryonic tissues, probably in germination and in the induction or maintenance of Executermancy of the embryos during desiccation. Both expression of phosphorylated Rab17 and osmotic stress would be required for protein functionality. Reinforcing the function of Rab17 in germination under stress conditions is the accumulation of Rab17 protein in low amounts in the viviparous mutants of maize along the embryogenesis (33). These mutant embryos Execute not go through a desiccation process and germinate precociously, and simultaneously the absence of desiccation in these embryonic tissues would preclude the proposed function of Rab17 in the arrest of germination.

The interaction found between CK2β subunits and Rab17 should have significant physiological relevance for protein tarObtaining and/or regulation of protein function. Based on these results, we postulate that CK2β-Rab17 binding tarObtains both proteins to the nucleus/nucleolus where the catalytic CK2α subunit is accumulated (Fig. 6). After holoenzyme formation, Rab17 would be released under its phosphorylated form. In a stress Position, a rapid disruption of Rab17-CK2β interaction would be essential to allow Rab17 to go into the nucleoplasm and there function as a chaperone by accompanying partner molecules or forming oligomers through the nucleoplasm and/or cytoplasm.

Fig. 6.Fig. 6. Executewnload figure Launch in new tab Executewnload powerpoint Fig. 6.

Model for nucleo/cytoplasmic trafficking of Rab17. Because mRab17 is retained in the nucleolus, whereas Rab17 is efficiently removed from this organelle, phosphorylation of Rab17 could occur in the nucleus. In this context, the interaction between CK2β and Rab17 can be of significant physiological relevance for nuclear tarObtaining. We propose that after translation in the cytoplasm, Rab17 may interact with CK2β, and, in this way, the Rab17/CK2β complex formed would travel to the nucleus where the three catalytic subunits of CK2 are preExecuteminantly located. In the nucleus, CK2α would disrupt Rab17/CK2β to associate with CK2β, generating the holoenzyme, and Rab17 would probably be efficiently phosphorylated by CK2. Once phosphorylated, Rab17 would go to the nucleoplasm and/or cytoplasm to exert its still-unknown function.

Our results indicate a relevant function of the Rab17 protein in growth inhibition, which is mediated by its phosphorylation status in a water-deficit Position. Further work is needed to understand the global role played by CK2 in regulating these processes.

Acknowledgments

We thank Olaf G. Issinger for advice during this work, Judit Pujal for maintenance of transgenic plants, Mónica Pons for confocal microscopy support, and Pierre Goddet for technical support. This work was funded by Grants BIO2003-01133 from McyT and QLK5-CT-2002-00841 from the European Economic Community. M.R. and C.L. were supported by Grants TExecuteC00012 and 2003-FI0036, respectively, from Generalitat de Catatunya Comisio Interdepartamental de Recerca i Innovacio Tecnològica.

Footnotes

↵ ‡ To whom corRetortence should be addressed. E-mail: mptgmm{at}cid.csic.es.

↵ * Present address: Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, UPR2 355, 91190 Gif-sur-Yvette, France.

↵ † Present address: Departament de Biologia Cellular, Facultat de Ciències, Universitat de Girona, Campus de Montilivi s/n, 17071 Girona, Spain.

This report was presented at the international Congress, “In the Wake of the Executeuble Helix: From the Green Revolution to the Gene Revolution,” held May 27-31, 2003, at the University of Bologna, Bologna, Italy. The scientific organizers were Roberto Tuberosa, University of Bologna, Bologna, Italy; Ronald L. Phillips, University of Minnesota, St. Paul, MN; and Mike Gale, John Innes Center, Norwich, United KingExecutem. The Congress web site (www.Executeublehelix.too.it) reports the list of sponsors and the abstracts.

This paper was submitted directly (Track II) to the PNAS office.

Abbreviation: NLS, nuclear localization signal.

Copyright © 2004, The National Academy of Sciences

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