Tyrosine Phosphorylation Based Homo-dimerization of Arabidopsis RACK1A Proteins Regulates Oxidative Stress Signaling Pathways in Yeast

Abstract

Scaffold proteins are known as important cellular regulators that can interact with multiple proteins to modulate diverse signal transduction pathways. RACK1 (Receptor for Activated C Kinase 1) is a WD-40 type scaffold protein, conserved in eukaryotes, from Chlamydymonas to plants and humans, plays regulatory roles in diverse signal transduction and stress response pathways. RACK1 in humans has been implicated in myriads of neuropathological diseases including Alzheimer and alcohol addictions. Model plant Arabidopsis thaliana genome maintains three different RACK1 genes termed RACK1A, RACK1B, and RACK1C with a very high (85-93%) sequence identity among them. Loss of function mutation in Arabidopsis indicates that RACK1 proteins regulate diverse environmental stress signaling pathways including drought and salt stress resistance pathway. Recently deduced crystal structure of Arabidopsis RACK1A- very first among all of the RACK1 proteins, indicates that it can potentially be regulated by post-translational modifications, like tyrosine phosphorylations and sumoylation at key residues. Here we show evidence that RACK1A proteins, depending on diverse environmental stresses, are tyrosine phosphorylated. Utilizing site-directed mutagenesis of key tyrosine residues, it is found that tyrosine phosphorylation can potentially dictate the homo-dimerization of RACK1A proteins. The homo-dimerized RACK1A proteins play a role in providing UV-B induced oxidative stress resistance. It is proposed that RACK1A proteins ability to function as scaffold protein may potentially be regulated by the homo-dimerized RACK1A proteins to mediate diverse stress signaling pathways.

Keywords: Arabidopsis; RACK1A; UV-B; homo-dimerization; oxidative stress; split-ubiquitin; yeast.

FIGURE 1

RACK1 proteins are capable of interacting with each other as shown by the proximity sensor- Split ubiquitin based assay. Full-length cDNAs from RACK1A, and from Y248F-RACK1A were used to construct the baits and preys. (A) Wild type bait and prey maintaining yeast cells are termed as AA and mutant bait and prey maintaining yeast cells are termed as YY. Growth of AA but not YY on FOA (5-fluoroorotic acid) containing plates confirm interaction. Known RACK1A interactor Plastocyanin (AT1G20340) is used as positive control (RACK1A bait and plastocyanin as prey (PA); while mutant Y248F-RACK1A fails to interact with wildtype plastocyanin (PY). (B) Same AA, YY, PY, and PA cells were grown on selective plates without uracil supplement. Interacting proteins (AA and PA) fails to grow on the plate as the URA3 reporter is degraded upon interaction; whereas YY and PY could grow as non-interaction allows to have the reporter URA3 intact that allows them to grow even without uracil supplement.

FIGURE 2

Stable expression of bait and prey constructs. (A) PCR amplification of bait construct using RACK1A and bait specific (lane 1- AA; lane 2- YY) or RACK1A and prey specific (lane 3- AA and lane 4- YY) primers. The empty vector containing EE cells did not show any amplification from either primer pairs (lane 5 and lane 6). No template control (lane 7) did not amplify any band. (B) Yeast cells maintaining both the bait and prey constructs [pMKZ-RACK1A:NUI-RACK1A (abbreviated as AA) and pMKZ- RACK1A-Y248F:NUI-RACK1A-Y248F (abbreviated as YY)] are able to grow of media lacking both the selection markers (minus histidine and tryptophan)- confirming stable expression of the constructs in the AA and YY yeast cells. (C) Serial dilution of the individual prey constructs (pMKZ-RACK1A (left upper) and pMKZ-RACK1A-Y248F (left lower) are able to grow on the SD-H selection plates while the individual bait constructs (NUI-RACK1A (right upper) and NUI-RACK1A-Y248F (right lower) are able to grow on the bait specific selection plates (SD-T).

FIGURE 3

BiFC assays confirm RACK1A homo-dimerization in vivo. BiFC analysis shows homo-dimerization of RACK1A in the nucleus and plasma membrane of onion epidermal strips. Onion epidermal cells bombarded with nYFP-RACK1A and cYFP-RACK1A constructs (AA) shows YFP fluorescence through fluorescence complementation while cells bombarded with the nYFP-Y248F-RACK1A and cYFP-Y248F-RACK1A (YY) constructs failed to show any fluorescence complementation.

FIGURE 4

Physiological effect of RACK1A homo-dimerization. (A) Phosphorylation of RACK1A Y248 residue under heat stress. Anti-pY248-RACK1A antibody is raised by adsorbing against the RACK1B and RACK1C peptides to ensure that the antibody would only recognize the Y248 phosphorylated RACK1A protein (Creative Diagnostics, Shirley, NY, USA). Y248 phosphorylation is enhanced in the presence of high heat (37°C) compared to the ambient heat (22°C). Arabidopsis plants (3 weeks-old) were incubated for 3 h at the indicated temperatures with constant light and as loading control, the ponceau stained blot image is presented at the bottom of the panel. (B) Stable expression of RACK1A protein is evident under the UV-B exposed (45 min) AA cells but UV-B exposed YY cells show instable RACK1A expression. Full length anti-RACK1A antibody detects both the RACK1A proteins in the bait and in the prey vectors. The lysates from the rack1a-1 knock-out Arabidopsis plants were used as negative control which did not show any 37 kD band but did show the Rubisco large subunit (RbcL ∼55 kD) band implying loading of lysates in the lane. Bands from non-specific binding lanes are used as loading control (lower lane). Note that Arabidopsis lysate did not show the non-specific band. The image is acquired using a ChemiDoc^TM XRS⁺ gel documentation system (Biorad, Hercules, CA, USA). (C) AA yeast cells can withstand UV-B induced stress conditions. AA and YY yeast cells were grown on M-HT medium at 30°C overnight, then exposed to UV-B lights (2.0 mW/cm²) for 45 min. As control, another set were treated to ambient light condition. An equal aliquot of the UV-B treated and non-treated cells were grown in M-HT medium for 48 h at 30°C. Yeast cell death is evaluated with the yeast viability kit (Invitrogen Inc. CA). AA cells show increased resistance to UV-B radiation induced cell death, whereas YY cells show susceptible phenotype (right). The non-treated AA and YY cells did not show significant cell death (left). The red lines above the + panels represents relative level of cell survival. (D) Quantitative evaluation of cell death under UV-B stress. Viable cells from three independent experiments as described in panel C were counted (total 300 cells). The mean is presented in the graph. The standard error of the mean from each treatment is also computed and is presented as bar on each mean value. (E) UV-B treated and non-treated AA and YY cells as in panel C were serial diluted to measure the growth potential on M-HT plates. AA cells as opposed to the YY cells show resistance to growth inhibition (lower). Non-treated AA and YY cells did not show any growth inhibition (upper).