Protein Kinase A Activity Is Necessary for Fission and Fusion of Golgi to Endoplasmic Reticulum Retrograde Tubules

<div><p>It is becoming increasingly accepted that together with vesicles, tubules play a major role in the transfer of cargo between different cellular compartments. In contrast to our understanding of the molecular mechanisms of vesicular transport, little is known about tubular transport. How signal transduction molecules regulate these two modes of membrane transport processes is also poorly understood. In this study we investigated whether protein kinase A (PKA) activity regulates the retrograde, tubular transport of Golgi matrix proteins from the Golgi to the endoplasmic reticulum (ER). We found that Golgi-to-ER retrograde transport of the Golgi matrix proteins giantin, GM130, GRASP55, GRASP65, and p115 was impaired in the presence of PKA inhibitors. In addition, we unexpectedly found accumulation of tubules containing both Golgi matrix proteins and resident Golgi transmembrane proteins. These tubules were still attached to the Golgi and were highly dynamic. Our data suggest that both fission and fusion of retrograde tubules are mechanisms regulated by PKA activity.</p></div

PKA activity in NRK cells is less sensitive to H-89 than in HeLa cells.

<p>NRK cells (A) or HeLa cells (B) were left untreated or treated with H-89 at the indicated concentrations for 60 min. Cell extracts were prepared to assess PKA activity using a non-radioactive protein kinase assay kit (Calbiochem), according to the manufacturer's instructions. PKA activity at each concentration of H-89 represents the percentage compared to the activity in untreated cells. N = 3.</p

Dynamic behavior of GFP-GRASP55 in accumulated retrograde tubules.

<p>NRK cells transiently expressing GFP-GRASP55 were held in a microscope stage at 37°C, and were treated with 5 μg/ml BFA in conjunction with 100 μM myr-PKI-A (A; n = 10) or 30 μM H-89 (B; n = 15). Live cells were examined by fluorescence microscopy, and the time after initiation of each treatment is shown in the upper right corner of each panel in minutes:seconds. In A, arrowheads indicate localization of GFP-GRASP55 at ER exit sites that during the treatment seems to vanish; large arrows indicate the elongation of a tubule (21:00–21:30) that retracts and dilates at a central portion (37:10), and the dilated part remains steady until the end of imaging (51:00); and small arrows indicate a small tubule that elongates at a later time (37:10). In B, small arrows indicate elongation of tubules at different times (19:50, 22:00, 22:50, 26:10, and 33:30), the curving and retraction of a tubule (23:30 to 24:50), the elongation and retraction of a tubule from the central portion of an accumulated tubule (26:10 to 28:10), and the elongation, curving, and retraction of another tubule (33:30 to 34:20); and arrowheads indicate the dilated tip of an accumulated tubule. Bars, 5 μm.</p

Dynamic behavior of GalT-YFP in accumulated retrograde tubules.

<p>NRK cells transiently expressing GalT-YFP were held in a microscope stage at 37°C, and were treated with 5 μg/ml BFA in conjunction with 100 μM myr-PKI-A (A; n = 10) or 30 μM H-89 (B; n = 15). Live cells were examined by fluorescence microscopy, and the time after initiation of each treatment is shown in the upper right corner of each panel in minutes:seconds. In A, large arrows indicate the dwindling of an initial thick tubule that leaves a swollen central portion; and small arrows indicate the formation of a dilated tip. In B, arrowheads indicate the dilated tip of an accumulated tubule; large arrows indicate a tubule that elongates and retracts; and small arrows indicate the elongation and retraction of another tubule. Bars, 5 μm.</p

P115 and GM130 localizes distinctly in accumulated retrograde tubules.

<p>NRK cells were left untreated (A) or treated with 5 μg/ml BFA (B), or with BFA in conjunction with 100 μM myr-PKI-A (C), for the time indicated. Cells were fixed, permeabilized, immunolabeled with rabbit polyclonal antibody to p115 and mouse monoclonal antibody to GM130, followed by Alexa-594-conjugated donkey anti-rabbit IgG (red channel), and Alexa-488-conjugated donkey anti-mouse IgG (green channel). Nuclei were stained with DAPI (blue channel). Stained cells were examined by fluorescence microscopy. Merging of the images in the red, green, and blue channels generated the third image on each row; yellow indicates overlapping localization of the green and red channels. In C, arrows indicate colocalization in accumulated tubules. Bar, 10 μm.</p

PKA activity is necessary for the fission of accumulated tubules.

<p>(A) NRK cells were treated with 5 μg/ml BFA in conjunction with 100 μM myr-PKI-A (m-PKI-A). After 60 min, myr-PKI-A was washed out in the presence of BFA, and cells were fixed at the times indicated. Cells were permeabilized and immunolabeled with rabbit polyclonal antibody to GRASP65, followed by Alexa-488-conjugated donkey anti-rabbit IgG. Stained cells were examined by fluorescence microscopy. Large arrows indicate large cut tubules. Small arrows indicate small cut tubules. Bar, 10 μm. (B) NRK cells transiently expressing GFP-GRASP55 were held in a microscope stage at 37°C. Cells were treated with 5 μg/ml BFA in conjunction with 30 μM H-89, and H-89 was washed out in the presence of BFA (n = 7). The large arrow indicates the site where an accumulated tubule is cut; small arrows indicate a punctum where the cut tubule collapses. Bar, 5 μm. (C) NRK cells transiently co-expressing CFP-GM130 and Sec13-YFP were held in a microscope stage at 37°C. Cells were treated, and myr-PKI-A was washed out as in A (n = 5). Arrows in the upper panels indicate the collapsing of a cut tubule into an ER exit site indicated by a large arrow. Bar, 5 μm. In B and C, live cells were examined by fluorescence microscopy, and the time after initiation of the treatment is shown in the upper or lower right corner of each panel, respectively, in minutes:seconds.</p

The tip of an accumulated tubule containing GM130 colocalizes with an ER exit site.

<p>NRK cells were left untreated (A) or treated with 5 μg/ml BFA (B), or with BFA in conjunction with 100 μM myr-PKI-A (C), for the time indicated. Cells were fixed, permeabilized, immunolabeled with mouse monoclonal antibody to GM130 and rabbit antibody to Sec23A, followed by Alexa-594-conjugated donkey anti-mouse IgG (red channel), and Alexa-488-conjugated donkey anti-rabbit IgG (green channel). Nuclei were stained with DAPI (blue channel). Stained cells were examined by confocal fluorescence microscopy. Merging of the images in the red, green, and blue channels generated the third image on each row; yellow indicates overlapping localization of the green and red channels. In B, arrows indicate colocalization at ER exit sites. In C, the arrow indicates colocalization of the tip of a tubule containing GM130 with an ER exit site. Bar, 10 μm.</p

Retrograde tubules accumulate upon PKA inhibition.

<p>NRK cells were left untreated (A) or treated with 5 μg/ml BFA (B-C), or with BFA in conjunction with either 30 μM H-89 (D) or 100 μM myr-PKI-A (E), for the time indicated. Cells were fixed, permeabilized, immunolabeled with mouse monoclonal antibody to GM130 and rabbit antibody to α-mannosidase II (Man-II), followed by Alexa-594-conjugated donkey anti-mouse IgG (red channel), and Alexa-488-conjugated donkey anti-rabbit IgG (green channel). Nuclei were stained with DAPI (blue channel). Stained cells were examined by confocal fluorescence microscopy. Merging of the images in the red, green, and blue channels generated the third image on each row; yellow indicates overlapping localization of the green and red channels. In B, arrowheads indicate the emergence of tubules containing GM130, but devoid of Man-II. In C, arrows indicate colocalization at ER exit sites. In D and E, arrowheads indicate colocalization at accumulated tubules, and arrows indicate colocalization at swollen portions of tubules. Bar, 10 μm.</p

Functional disruption of the Golgi apparatus protein ARF1 sensitizes MDA-MB-231 breast cancer cells to the antitumor drugs Actinomycin D and Vinblastine through ERK and AKT signaling

<div><p>Increasing evidence indicates that the Golgi apparatus plays active roles in cancer, but a comprehensive understanding of its functions in the oncogenic transformation has not yet emerged. At the same time, the Golgi is becoming well recognized as a hub that integrates its functions of protein and lipid biosynthesis to signal transduction for cell proliferation and migration in cancer cells. Nevertheless, the active function of the Golgi apparatus in cancer cells has not been fully evaluated as a target for combined treatment. Here, we analyzed the effect of perturbing the Golgi apparatus on the sensitivity of the MDA-MB-231 breast cancer cell line to the drugs Actinomycin D and Vinblastine. We disrupted the function of ARF1, a protein necessary for the homeostasis of the Golgi apparatus. We found that the expression of the ARF1-Q71L mutant increased the sensitivity of MDA-MB-231 cells to both Actinomycin D and Vinblastine, resulting in decreased cell proliferation and cell migration, as well as in increased apoptosis. Likewise, the combined treatment of cells with Actinomycin D or Vinblastine and Brefeldin A or Golgicide A, two disrupting agents of the ARF1 function, resulted in similar effects on cell proliferation, cell migration and apoptosis. Interestingly, each combined treatment had distinct effects on ERK1/2 and AKT signaling, as indicated by the decreased levels of either phospho-ERK1/2 or phospho-AKT. Our results suggest that disruption of Golgi function could be used as a strategy for the sensitization of cancer cells to chemotherapy.</p></div

Effect of the combined treatment of Golgicide A with Actinomycin D or Vinblastine on the levels of phospho-ERK1/2 and phospho-AKT in MDA-MB-231 cells.

<p>(A-B) Cells were left untreated for 5 h (<i>Control</i>), or treated 5 h either with 10 μM Golgicide A (<i>GCA</i>), 10 ng/ml Actinomycin D (<i>ActD</i>; A) or 25 nM Vinblastine (<i>VLB</i>; B), or with GCA in conjunction either with ActD (<i>GCA + ActD</i>; A) or VLB (<i>GCA + VLB</i>; B). After solubilizing in detergent, proteins were analyzed by SDS-PAGE followed by immunoblotting using antibodies to the proteins indicated on the right. The position of molecular mass markers is indicated on the left. (C-D) Densitometry quantification of the immunoblot signal of the levels of phospho-ERK1/2 as shown in <i>A</i> and <i>B</i> (C), and of the levels of phospho-AKT as shown in <i>A</i> and <i>B</i> (D). Bar represents the mean ± standard deviation (n = 3). * <i>P</i> < 0.05; ** <i>P</i> < 0.01; *** <i>P</i> < 0.001; ns, not statistically significant.</p