Protein kinase CK2 modulates developmental functions of the abscisic acid responsive protein Rab17 from maize

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 show the specific interaction of Rab17 with the CK2 regulatory subunits CK2 beta-1 and CK2 beta-3, and that these interactions do 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 CK2 beta subunits and Rab17 distributed over the cytoplasm and nucleus. By contrast, catalytic CK2 alpha subunits and a Rab17 mutant protein (mRab17) that is not a substrate for CK2 phosphorylation remain accumulated in the nucleoli. A dual-color image shows 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.

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).

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.

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.

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 underneath 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 endogenous 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 underneath for each lane.

Fig. 5.

Overexpression of Rab17 and mRab17 in Arabidopsis thaliana. (A Top) Western blot of protein extracts from leaves from Arabidopsis transgenic plants using the anti-Rab17 antibody. Samples correspond 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 open 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) Differences in radicle emergence (days 4-6 of germination) and cotyledon expansion (day 11 of germination) in Rab17 and mRab17 transgenic lines.

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 targeting. 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 predominantly 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.