Arabidopsis casein kinase 1-like 6 contains a microtubule-binding domain and affects the organization of cortical microtubules,

Abstract

Members of the casein kinase 1 (CK1) family are evolutionarily conserved eukaryotic protein kinases that are involved in various cellular, physiological, and developmental processes in yeast and metazoans, but the biological roles of CK1 members in plants are not well understood. Here, we report that an Arabidopsis (Arabidopsis thaliana) CK1 member named casein kinase 1-like 6 (CKL6) associates with cortical microtubules in vivo and phosphorylates tubulins in vitro. The unique C-terminal domain of CKL6 was shown to contain the signal that allows localization of CKL6 to the cortical microtubules. This domain on its own was sufficient to associate with microtubules in vivo and to bind tubulins in vitro. CKL6 was able to phosphorylate soluble tubulins as well as microtubule polymers, and its endogenous activity was found to associate with a tubulin-enriched subcellular fraction. Two major in vitro phosphorylation sites were mapped to serine-413 and serine-420 of tubulin beta. Ectopic expression of wild-type CKL6 or a kinase-inactive mutant form induced alterations in cortical microtubule organization and anisotropic cell expansion. Collectively, these results demonstrate that CKL6 is a protein kinase containing a novel tubulin-binding domain and plays a role in anisotropic cell growth and shape formation in Arabidopsis through the regulation of microtubule organization, possibly through the phosphorylation of tubulins.

Figure 1.

Association with cortical microtubules by the CTD and CKL6 in vivo. A, Association of CKL6:GFP with cortical microtubules in epidermal cotyledon cells of a transgenic Arabidopsis seedling and in transgenic tobacco BY-2 cells. Confocal images were taken from the green channel to detect fluorescent signals produced by GFP-tagged CKL6. Bars = 20 μm. B, Cytoskeletal patterns exhibited by GFP:CTD in the cotyledon (left) or hypocotyl (right) epidermal cells of transgenic Arabidopsis seedlings at 7 d after germination. C, AMP treatment shows the disruption of cortical microtubules that are labeled by GFP:Tua (top) or GFP:CTD (bottom). Images were taken at 5 and 30 min of incubation after application of 100 μm APM. Bars = 10 μm.

Figure 2.

The CTD contains a subdomain important for the association with microtubules (MT). A series of deletion mutants derived from the CTD was produced to map the microtubule-associating subdomain within the CTD. Microtubule association of GFP-tagged CTD mutants was assessed by localization studies in epidermal leaf cells of Arabidopsis. Representative images are shown to support the qualitative analysis summarized in the diagram. All images are three-dimensional images reconstructed from a series of optical Z-sections. A, A diagram illustrating a series of CTD deletion mutants and the extent of their microtubule association, as qualitatively determined in comparison with the full-length CTD. AA, Amino acids. B, GFP:CTD_302–479 (full length). C, GFP:CTD_351–479. D, GFP:CTD_302–396. E, GFP:CTD_397–479.

Figure 3.

In vitro interaction of CTD and CKL6 with tubulins. A, Control pull-down assays were performed using 1 μg of GST immobilized on glutathione-agarose beads as bait per assay (top). Fractions containing each prey protein bound (B) or unbound (U) to GST were separated on 12% SDS-PAGE gels and stained with Coomassie Brilliant Blue. Prey molecules examined included bovine tubulin dimers (Tub αβ), recombinant Arabidopsis tubulin α6 and β3 fused to MBP (M-Tubα and M-Tubβ, respectively), and MBP. One microgram of prey protein was used in each assay. White circles indicate the size of each prey protein expected to be detected in the bound fraction if pulled down by the bead-bound GST. Pull-down assays employing 1 μg of GST:CTD as bait show specific interaction between GST:CTD and tubulins (bottom). White asterisks indicate the prey molecules that are bound to GST:CTD. B, Pull-down assays performed employing GST or GST:CKL6 as prey for M-Tubα, M-Tubβ, or MBP. The bait proteins (1 μg each) were immobilized on maltose-agarose beads and incubated with 1 μg of prey proteins. Bound and unbound fractions were collected followed by separation on 12% SDS-PAGE gels and electroblotting onto nitrocellulose membranes. GST or GST:CKL6 in each fraction was detected by specific antibodies to GST.

Figure 4.

Phosphorylation of tubulins by CKL6. A, Phosphorylation of bovine tubulin dimers (Tubαβ) by CKL6. Phosphorylation of TMV MP (TMP) was included as a positive control. B, Phosphorylation of recombinant tubulin monomers. C, Phosphorylation of microtubule polymers (MT) by CKL6. Phosphorylation reactions were centrifuged at 100,000g for 15 min to separate soluble (S) fraction from the pellet (P) fraction. Soluble tubulin dimers and bovine serum albumin (BSA) were included as positive and negative controls for the phosphorylation and sedimentation, respectively.

Figure 5.

In vitro phosphorylation site mapping for tubulin β3. A, A schematic diagram illustrating the positions of substitution and truncation mutants of Arabidopsis tubulin β3. B, Phosphorylation assays employing the recombinant tubulin β3 mutants fused to MBP as substrates for CKL6. C, Amino acid sequence alignment of tubulin β isoforms from Arabidopsis and bovine at the C-terminal end, showing the conserved phosphorylation sites Ser-413 and Ser-420 (asterisks) within helix 12.

Figure 6.

CKL6 activity contained within the tubulin/MAPs-enriched subcellular fraction. A, Western analysis showing an accumulation of CKL6 within the tubulin/MAPs-enriched subcellular fraction. Western blotting was performed employing CKL6-specific antibodies (α-CKL6) or tubulin-specific antibodies (α-Tub) against the solubilized tubulin/MAPs-enriched fraction. Recombinant GST:CKL-6 was included as a positive control for α-CKL6. Lane P, Protein profile of the tubulin/MAPs fraction separated on a 10% SDS-PAGE gel and stained with Coomassie Brilliant Blue. B, Phosphorylation assays were performed in reactions employing the solubilized tubulin/MAPs fraction as a kinase source and recombinant tubulins α and β (M-Tubα and M-Tubβ, respectively), bovine tubulin dimers (Tubαβ), phospho-mutant S_413/420→A_413/420 (A413/420), and TMV MP. White asterisks mark the position of each substrate.

Figure 7.

Cellular phenotypes induced by overexpression of CKL6 and CKL6-KI. A to C, Confocal images showing the cortical microtubule patterns labeled with ectopically expressed GFP:Tua in epidermal leaf cells of 6-d-old Arabidopsis seedlings. Z-series of 1-μm-thick optical sections were captured and projected as three-dimensional images. A, Control pavement cells. B, Pavement cells of an F1 seedling expressing CKL6 and GFP:Tua. C, Pavement cells of an F1 seedling expressing CKL6-KI and GFP:Tua. Arrowheads indicate the abnormal array patterns of the microtubules. Bars = 25 μm. D to E, Confocal images of a single optical section showing the shapes of epidermal cells. Expanded areas including the cells shown in A to C are presented. D, Control epidermal cells. E, Epidermal cells of an F1 seedling expressing CKL6 and GFP:Tua. F, Epidermal cells of an F1 seedling expressing CKL6-KI and GFP:Tua. Bars = 25 μm.