Phosphorylation of p23-1 cochaperone by protein kinase CK2 affects root development in Arabidopsis

Abstract

Root growth is a fundamental process in plants and assures nutrient and water uptake required for efficient photosynthesis and metabolism. Postembryonic development of roots is controlled by the functionality of the meristem. Several hormones and signaling molecules regulate the size of the meristem, and among them, auxins play a major role. Protein kinase CK2, along with the chaperone protein HSP90, has been found to be involved in the regulation of auxin transport. Here, we show that p23-1, a cochaperone of HSP90, is phosphorylated by CK2 in Arabidopsis. We identified Ser201 as the major CK2 target site in p23-1 and demonstrated that phosphorylation of this site is necessary for normal root development. Moreover, we shed light on the nature of CK2 in Arabidopsis, showing that the three catalytic isoforms, CK2 αA, αB and αC, are proteins of approximately 40 kDa. Our results increase knowledge of the connection among HSP90, p23-1 and CK2 in Arabidopsis, suggesting the existence of a possible common root development mechanism controlled by these signaling molecules.

Figure 1

p23-1 phosphorylation by CK2-like activity. (A) Representative autoradiography (left) and Coomassie staining (right) after radioactive phosphorylation of increasing concentrations of recombinant p23-1 and p23-2, as indicated, by 10 μg of Arabidopsis total protein extract and separation via SDS-PAGE. The arrows indicate the migrations of each p23 isoform. Equally labeled radioactive bands in all lanes are due to autophosphorylation of the proteins present in the extract (see lane 1, where no p23 was present). The migration of the most abundant protein rubisco (~55 kDa) is also indicated. (B) Kinetics showing phosphorylation by CK2 with increasing concentrations of p23-1 and p23-2. The calculated kinetics values are shown in the box. Vmax is reported as pmol/min/mg, Km as μM. Quantification was performed by excising bands from the gel, as shown in panel A, and scintillator counting. Values are the means ± SD of independent experiments. (C) p23-1 (0.1 μg) was phosphorylated by 10 μg of Arabidopsis total protein extract in the presence (as indicated) of 2 μM TBB (TBB), 10 nM CX4945 (CX), or 1 μM Staurosporin (ST), or DMSO solvent as the control (CTR). Representative autoradiography is shown after protein separation by SDS-PAGE. The arrow indicates the migration of p23-1. Mw markers migrations are also shown on the left.

Figure 2

p23-1 phosphorylation by a monomeric 40-kDa CK2. (A) p23-1 (0.1 μg, lanes 1 and 2) was incubated with recombinant human monomeric CK2 (α, 15 ng) or tetrameric CK2 (α₂β₂, 5 ng); β-casein (1 μg, lanes 3 and 4) was used to ensure that the amount of each CK2 isoform chosen had the same catalytic activity towards a model substrate. After radioactive phosphorylation (10 min at 30 °C), samples were resolved by SDS-PAGE. A representative autoradiograph is shown. (B) Representative autoradiography of an in-gel kinase assay: 10 or 20 μg protein from Arabidopsis total extract (in duplicate, as indicated) was resolved by SDS-PAGE in which p23-1 (10 μg/ml) was included in the gel. In lane 1, human recombinant CK2 α (hsCK2α 50 ng, Mw 40 kDa) was loaded as a positive control. After electrophoresis and protein renaturation, the gel was incubated with a radioactive phosphorylation mixture and analyzed by autoradiography. The migration of Mw markers is shown on the left.

Figure 3

p23-1 phosphorylation activity of Arabidopsis mutant lines. (A) The specific CK2 peptide substrate CK2-tide was incubated with 10 μg total extract proteins from wild type (wt) or the CK2 αC mutant (α3), CK2 αAαB mutant (αAαB) or of CK2 αAαBαC (triple) mutant lines in the presence of a radioactive phosphorylation mixture. Blank controls were performed in the presence of 10 nM CX4945. Each phosphorylation activity is reported after subtraction of the relative blank control. (B) p23-1 wt or the S201A mutant (0.1 μg; lane #6) was incubated with 10 μg total extract proteins from wild type (wt), CK2 αC mutant (αC), CK2 αAαB mutant (αAαB) or the CK2 αAαBαC (triple) mutant, as indicated. CX4945 (10 nM) was present where indicated. A representative autoradiograph after protein separation by SDS-PAGE is shown. The migration of Mw markers is shown on the left. (C) Quantification of p23-1 radioactivity is shown, obtained by Cyclone Plus Storage Phosphor System (PerkinElmer) analysis from the gel of panel B.

Figure 4

Arabidopsis CK2 nuclear catalytic isoform expression. (A) Expression levels of CK2 αA, CK2 αB or CK2 αC assayed by quantitative Real-Time PCR of the 3′-region of the mRNA. Values are reported as the percentage of the Actin 2 (ACT2) expression level in the shoot (white bars) or root (black bars) of 10-day-old Arabidopsis seedlings. (B) Expression levels of CK2 αA, CK2 αB or CK2 αC assayed by quantitative Real-Time PCR on the 5′-region of the mRNA. Values are reported as the percentage of the Actin 2 (ACT2) expression level in the shoot (white bars) or root (black bars) of 10-day-old Arabidopsis seedlings.

Figure 5

Impact of p23 phosphorylation on primary root development. (A) Propidium iodide stained roots of 10-day-old seedlings from the various lines: wild type (wt), single mutant (p23-1), complemented line (p23-1 35 S:p23-1), phospho-mimetic line (p23-1 35 S:p23-1-S201E), complemented mutant line (p23-1 35 S:p23-1-S201A). The excitation and emission wavelengths for confocal acquisition were 488 nm and 600–650 nm, respectively. The arrows show the upper limit of the meristematic zone. Scale bar = 50 μm. (B) The meristem cell number in the different genetic backgrounds. (^aP < 0.01 compared with the wt, ^bP < 0.01 compared with the background line; both Student’s t-test).