Tumor necrosis factor induces hyperphosphorylation of kinesin light chain and inhibits kinesin-mediated transport of mitochondria - PubMed (original) (raw)

Tumor necrosis factor induces hyperphosphorylation of kinesin light chain and inhibits kinesin-mediated transport of mitochondria

K De Vos et al. J Cell Biol. 2000.

Abstract

The molecular motor kinesin is an ATPase that mediates plus end-directed transport of organelles along microtubules. Although the biochemical properties of kinesin are extensively studied, conclusive data on regulation of kinesin-mediated transport are largely lacking. Previously, we showed that the proinflammatory cytokine tumor necrosis factor induces perinuclear clustering of mitochondria. Here, we show that tumor necrosis factor impairs kinesin motor activity and hyperphosphorylates kinesin light chain through activation of two putative kinesin light chain kinases. Inactivation of kinesin, hyperphosphorylation of kinesin light chain, and perinuclear clustering of mitochondria exhibit the same p38 mitogen-activated kinase dependence, indicating their functional relationship. These data provide evidence for direct regulation of kinesin-mediated organelle transport by extracellular stimuli via cytokine receptor signaling pathways.

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Figures

Figure 1

Figure 1

Inhibition of kinesin motor activity in TNF-treated L929 cells. A and B, MAP-depleted cytosol was prepared from untreated (CTRL) and TNF-treated (TNF) L929 cells and the level of kinesin motor activity in these preparations was tested in an MT-gliding assay (A) and a mitochondrial motility assay (B). In the MT-gliding assay, coverslips were coated with the respective cytosols, and rhodamine-labeled MTs were added together with ATP. The number of gliding MTs was determined relative to the total number of MTs present in a microscopic field. To identify the active molecular motor present in the untreated cytosol, SUK4 mAb was added (SUK4). Data shown are the mean and SEM calculated from at least ten different microscopic fields and are representative of a minimum of three independent experiments. The values of control and TNF samples were significantly different as determined using a one-tailed heteroscedastic t test (P < 0.01). In the mitochondrial motility assay, MTs were absorbed on coverslips and a reaction mix containing mitotracker-labeled mitochondria, ATP, and cytosol from untreated or TNF-treated L929 cells was added. The number of moving mitochondria per microscopic field was determined. The results shown represent the mean and SEM of 25 fields and are representative of three independent experiments (P < 0.01). C, The merged images of mitochondrial motility were generated by overlaying stacks of photos of mitochondria (green) collected at 4-s intervals with a photo of the immobilized MTs (red). The starting position of the mitochondria is indicated by the open arrow, the end position by the filled arrow. The plus end (+) and the minus end (–) of the MTs are indicated. D, The KHC and KLC content of the respective cytosols used in the gliding and mitochondrial motility assays was verified by Western blot with SUK4 mAb and antipan-KLC Ab, respectively. E, Mitochondria were purified from untreated (CTRL) and TNF-treated (TNF) L929 cells and the amount of copurifying KHC was determined by Western blot analysis using anti-KIF5B Ab.

Figure 1

Figure 1

Inhibition of kinesin motor activity in TNF-treated L929 cells. A and B, MAP-depleted cytosol was prepared from untreated (CTRL) and TNF-treated (TNF) L929 cells and the level of kinesin motor activity in these preparations was tested in an MT-gliding assay (A) and a mitochondrial motility assay (B). In the MT-gliding assay, coverslips were coated with the respective cytosols, and rhodamine-labeled MTs were added together with ATP. The number of gliding MTs was determined relative to the total number of MTs present in a microscopic field. To identify the active molecular motor present in the untreated cytosol, SUK4 mAb was added (SUK4). Data shown are the mean and SEM calculated from at least ten different microscopic fields and are representative of a minimum of three independent experiments. The values of control and TNF samples were significantly different as determined using a one-tailed heteroscedastic t test (P < 0.01). In the mitochondrial motility assay, MTs were absorbed on coverslips and a reaction mix containing mitotracker-labeled mitochondria, ATP, and cytosol from untreated or TNF-treated L929 cells was added. The number of moving mitochondria per microscopic field was determined. The results shown represent the mean and SEM of 25 fields and are representative of three independent experiments (P < 0.01). C, The merged images of mitochondrial motility were generated by overlaying stacks of photos of mitochondria (green) collected at 4-s intervals with a photo of the immobilized MTs (red). The starting position of the mitochondria is indicated by the open arrow, the end position by the filled arrow. The plus end (+) and the minus end (–) of the MTs are indicated. D, The KHC and KLC content of the respective cytosols used in the gliding and mitochondrial motility assays was verified by Western blot with SUK4 mAb and antipan-KLC Ab, respectively. E, Mitochondria were purified from untreated (CTRL) and TNF-treated (TNF) L929 cells and the amount of copurifying KHC was determined by Western blot analysis using anti-KIF5B Ab.

Figure 1

Figure 1

Inhibition of kinesin motor activity in TNF-treated L929 cells. A and B, MAP-depleted cytosol was prepared from untreated (CTRL) and TNF-treated (TNF) L929 cells and the level of kinesin motor activity in these preparations was tested in an MT-gliding assay (A) and a mitochondrial motility assay (B). In the MT-gliding assay, coverslips were coated with the respective cytosols, and rhodamine-labeled MTs were added together with ATP. The number of gliding MTs was determined relative to the total number of MTs present in a microscopic field. To identify the active molecular motor present in the untreated cytosol, SUK4 mAb was added (SUK4). Data shown are the mean and SEM calculated from at least ten different microscopic fields and are representative of a minimum of three independent experiments. The values of control and TNF samples were significantly different as determined using a one-tailed heteroscedastic t test (P < 0.01). In the mitochondrial motility assay, MTs were absorbed on coverslips and a reaction mix containing mitotracker-labeled mitochondria, ATP, and cytosol from untreated or TNF-treated L929 cells was added. The number of moving mitochondria per microscopic field was determined. The results shown represent the mean and SEM of 25 fields and are representative of three independent experiments (P < 0.01). C, The merged images of mitochondrial motility were generated by overlaying stacks of photos of mitochondria (green) collected at 4-s intervals with a photo of the immobilized MTs (red). The starting position of the mitochondria is indicated by the open arrow, the end position by the filled arrow. The plus end (+) and the minus end (–) of the MTs are indicated. D, The KHC and KLC content of the respective cytosols used in the gliding and mitochondrial motility assays was verified by Western blot with SUK4 mAb and antipan-KLC Ab, respectively. E, Mitochondria were purified from untreated (CTRL) and TNF-treated (TNF) L929 cells and the amount of copurifying KHC was determined by Western blot analysis using anti-KIF5B Ab.

Figure 2

Figure 2

TNF induces hyperphosphorylation of KLC. A and B, Total lysates of untreated (CTRL) and TNF-treated (TNF) cells were separated by 2-DE (IP and MW) and transferred to PVDF membranes for Western blot analysis. KLC (A) and KHC (B) were revealed with antipan-KLC Ab and SUK4 mAb respectively. The filled arrow indicates isoelectric isoforms that disappear upon TNF-treatment. Open arrows indicate TNF-induced isoforms. C, KLC, coimmunoprecipitated with KHC (Immunop.) from untreated (CTRL) or TNF-treated (TNF) cells was incubated with γ[P32]-ATP alone (left). Alternatively, KLC coimmunoprecipitated with KHC from untreated L929 cells was supplemented with cytosol of untreated or TNF-treated cells (right). After completion of the in vitro kinase assay, the immune complex was separated by SDS-PAGE. P32 incorporation was visualized by PhosphorImager.

Figure 3

Figure 3

The p38MAPK inhibitor SB203580 inhibits TNF-induced KLC hyperphosphorylation. A, Total lysates of SB203580-treated (SB), and SB203580/TNF-cotreated (SB/TNF) L929 cells were separated by 2-DE, transferred to PVDF membranes, and were analyzed by Western blot with antipan-KLC Ab. B, KLC, coimmunoprecipitated with SUK4 mAb (Immunop.) from untreated (CTRL), SB203580-treated (SB), TNF-treated (TNF), or SB203580/TNF-cotreated (SB/TNF) L929 cells incubated with γ[P32]-ATP alone (left) or supplemented with cytosol of the respective cell populations (right). Next, the immune complex was separated by SDS-PAGE and P32 incorporation was visualized by PhosphorImager.

Figure 4

Figure 4

MT-gliding and motility of mitochondria are inhibited by SB203580. A and B, L929 cells were left untreated (CTRL), treated with SB203580 (SB) or TNF (TNF), or cotreated with SB203580 and TNF (SB/TNF). MAP-depleted cytosol was prepared and the activity of kinesin was tested in an MT-gliding assay (A) and a mitochondrial motility assay (B). *Values are not significantly different, as determined using a one-tailed heteroscedastic t test (P > 0.06). C, The KHC and KLC content of the respective cytosols used in the gliding and mitochondria motility assays was verified by Western blot with SUK4 mAb and antipan-KLC Ab, respectively.

Figure 5

Figure 5

Conventional kinesin transports mitochondria in L929 cells. A, L929 cells were left unloaded (CTRL), or were syringe-loaded with irrelevant Ab (Irr.) or SUK4 mAb (SUK4). After overnight recovery of the cells, the distribution of R123-stained mitochondria was analyzed by CLSM in the cells that had taken up Ab. The values shown represent the mean and SEM of four randomly chosen microscopic fields each containing ∼40 loaded cells. B, The SUK4 mAb-loaded cell typically shows a clustered distribution of mitochondria. In contrast, the irrelevant Ab-loaded cell retained the spread distribution of mitochondria. The line indicates the cell periphery.

Figure 6

Figure 6

TNF-induced clustering of mitochondria is inhibited by SB203580. The frequency of cells exhibiting the clustered mitochondrial phenotype was measured by CLSM of R123-stained mitochondria 4 h after stimulation. The values shown represent the mean and SEM of four randomly chosen microscopic fields each containing ∼60 cells. *Values are not significantly different as determined using a one-tailed heteroscedastic t test (P > 0.07).

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