Ikeda K et al. (2011), CK1 activates minus-end-directed transport of m...

XB-ART-42817

Mol Biol Cell 2011 Apr 15;228:1321-9. doi: 10.1091/mbc.E10-09-0741.

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CK1 activates minus-end-directed transport of membrane organelles along microtubules.

Ikeda K , Zhapparova O , Brodsky I , Semenova I , Tirnauer JS , Zaliapin I , Rodionov V .

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Microtubule (MT)-based organelle transport is driven by MT motor proteins that move cargoes toward MT minus-ends clustered in the cell center (dyneins) or plus-ends extended to the periphery (kinesins). Cells are able to rapidly switch the direction of transport in response to external cues, but the signaling events that control switching remain poorly understood. Here, we examined the signaling mechanism responsible for the rapid activation of dynein-dependent MT minus-end-directed pigment granule movement in Xenopus melanophores (pigment aggregation). We found that, along with the previously identified protein phosphatase 2A (PP2A), pigment aggregation signaling also involved casein kinase 1ε (CK1ε), that both enzymes were bound to pigment granules, and that their activities were increased during pigment aggregation. Furthermore we found that CK1ε functioned downstream of PP2A in the pigment aggregation signaling pathway. Finally, we discovered that stimulation of pigment aggregation increased phosphorylation of dynein intermediate chain (DIC) and that this increase was partially suppressed by CK1ε inhibition. We propose that signal transduction during pigment aggregation involves successive activation of PP2A and CK1ε and CK1ε-dependent phosphorylation of DIC, which stimulates dynein motor activity and increases minus-end-directed runs of pigment granules.

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Species referenced: Xenopus laevis
Genes referenced: camp csnk1a1 dnai1 krt8 mtnr1al ptpa

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	FIGURE 1:. CK1Îµ and PP2A are bound to pigment granules. (A) Immunoblots of cell extracts (left lanes) or preparations of pigment granules (right lanes) probed for CK1 isoforms (top) or the catalytic subunit of PP2A (bottom). PP2A and CK1Îµ, but not other CK1 isoforms, are enriched in preparations of purified pigment granules. (B) Fluorescence images of melanophores expressing GFP-CK1Îµ (top row) or stained with LysoTracker (bottom row) sequentially treated with melatonin (left column) and MSH (right column) to induce aggregation and dispersion of pigment granules. GFP-CK1Îµ is localized to fluorescent dots that accumulate in the cell center or redisperse throughout the cytoplasm after treatment with melatonin and MSH, respectively, as would be expected from pigment granules, whereas the distribution of lysosomes does not change significantly in response to hormones. Scale bar, 20 Î¼m.
	FIGURE 2:. CK1 activity is required for pigment granule aggregation and maintenance of the aggregated state. Quantification of responses of pigment granules to pigment aggregating or dispersing hormones melatonin (A and B) or MSH (C) in cells pretreated with various concentrations of a broad-specificity CK1 inhibitor D4476, or the CK1Î´ and CK1Îµ specific inhibitor IC261. (D) Responses of pigment granules to IC261 in cells with pigment aggregated with melatonin. Data are expressed as the percentage of cells with aggregated (white bars), partially aggregated (gray bars), or dispersed (black bars) granules. In panel B, the large fraction of cells with dispersed pigment granules among IC261-untreated melanophores (0 Î¼M) is explained by reduced rate of pigment aggregation in the presence of Taxol, which was used in this experiment to prevent MT depolymerization induced by IC261. D4476 and IC261 treatments inhibit melatonin-induced aggregation, but not MSH-induced dispersion of pigment granules; IC261 causes pigment dispersion in melatonin-treated cells with aggregated granules.
	FIGURE 3:. Enzymatic activities of the pigment granule-bound CK1 and PP2A are increased upon pigment aggregation. Activities of CK1 (A) or PP2A (B) measured in preparations of pigment granules isolated from melanophores stimulated with melatonin (Mel) or MSH, to aggregate or disperse pigment granules, respectively. The bar labeled Mel+OA (panel A) shows the data for melatonin-stimulated cells pretreated with the PP2A inhibitor okadaic acid. Average values determined for melatonin-treated cells are set at 100%. PP2A and CK1 activities are significantly higher in preparations of pigment granules obtained from melatonin- than MSH-stimulated cells, which shows that the activities of both enzymes increase during granule aggregation; okadaic acid treatment reduces the activity of CK1, indicating that CK1 activity is stimulated by PP2A.
	FIGURE 4:. Overexpression of a constitutively active CK1Îµ mutant partially rescues the inhibition of pigment granule aggregation by the PP2A inhibitor okadaic acid. (A) Quantification of response to melatonin of nontransfected melanophores, or melanophores expressing GFP, wild-type GFP-CK1Îµ, or the mutant GFP-CK1Îµ with constitutively high enzymatic activity; prior to stimulation with melatonin cells were treated with PP2A inhibitor okadaic acid (OA). Data are expressed as the percentage of cells with aggregated (white bars), partially aggregated (gray bars), or dispersed (black bars) pigment. OA treatment significantly inhibits aggregation of pigment granules in nontransfected melanophores and in cells expressing GFP or wild-type GFP-CK1Îµ. Expression of the constitutively active CK1Îµ mutant partially rescues OA-induced inhibition of pigment granule aggregation as evidenced from the increased fraction of cells with aggregated granules. (B) Comparison of the levels of expression of GFP and CK1Îµ constructs; wild-type and the constitutively active mutant CK1Îµ are expressed at approximately the same levels, whereas GFP is expressed at significantly higher levels compared with CK1Îµ constructs.
	FIGURE 5:. CK1 phosphorylates DIC in vivo and in vitro. (A) Immunoprecipitation of dynein with GFP antibody from extracts of GFP-DICâ€“expressing melanophores metabolically labeled with 32P. Left panel, Coomassie-stained gel; right panel, autoradiograph; MSH, Mel, and Mel+IC261 on top of each panel indicate extracts of cells treated with MSH (to induce pigment dispersion), melatonin (to induce aggregation), or IC261 and melatonin (to inhibit CK1 activity prior to induction of aggregation), respectively; M, lanes with molecular weight markers. In immunoprecipitates, 32P incorporates predominantly into GFP-DIC; this incorporation is increased in cells stimulated to aggregate pigment granules, and this increase is diminished by inhibition of CK1 activity. (B) In vitro phosphorylation by CK1 of dynein immunoprecipitated with an anti-GFP antibody from the extracts of GFP-DICâ€“expressing cells; left, Coomassie-stained gel of immunoprecipitate; right, autoradiograph of immunoprecipitate incubated with Î³-32P[ATP] in the presence (left) or absence (right) of recombinant CK1; DHC, GFP-DIC, antibody, and CK1 indicate positions of dynein heavy chain, GFP-DIC fusion protein, antibody heavy and light chains, and recombinant CK1, respectively. CK1 predominantly phosphorylates the protein with electrophoretic mobility similar to GFP-DIC.
	FIGURE 6:. Model for the activation of MT minus-endâdirected transport of pigment granules during pigment aggregation in melanophores. Melatonin decreases intracellular levels of cAMP, which in turn reduces the activity of PKA. PKA inactivation relieves inhibition of PP2A, which then dephosphorylates and activates CK1Îµ. CK1Îµ phosphorylates DIC, and this phosphorylation stimulates dynein activity and increases the length of MT minus-endâdirected runs of pigment granules, leading to their aggregation in the cell center.

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