IndraLab

"Phosphorylation of VCIP135 at S130 attenuates p97/p47-mediated Golgi membrane fusion."

"VCIP135 is phosphorylated on multiple sites at the C-terminus in addition to S130 (Totsukawa ; Zhang ), and a total of 13 sites on VCIP135 are phosphorylated in mitosis (Supplemental Figure 1)."

"The DUB activity was low when VCIP135 was highly phosphorylated at S130 in early mitosis (Figure 4C, lanes 2–4); while the DUB activity increased significantly when S130 was dephosphorylated in late mitosis (Figure 4C, lanes 5–7)."

"Because VCIP135 DUB activity is specifically required for postmitotic Golgi membrane fusion, we determined the effects of S130 phosphorylation of VCIP135 on postmitotic Golgi reassembly in vivo."

"Taken together, these results demonstrate that S130 phosphorylation negatively regulates VCIP135 activity, which controls Golgi structure formation during the cell cycle."

"We have developed a phospho-antibody that specifically recognizes phosphorylated S130 on VCIP135 (Figure 3). Biochemical (Figure 4) and microscopy (Figures 4 and 5) studies showed that S130 is phosphorylated from early mitosis to anaphase, which inactivates VCIP135 DUB activity."

"Mitosis-arrested cells were obtained by treatment with 100 ng/ml nocodazole (Sigma-Aldrich, St. Louis, MO) for 18 h. Mitotic-arrested cells were then washed with fresh medium without nocodazole five times; placed into new dishes; and incubated for another 0.5, 1, 1.5, 2, and 3 h. For determination of the effects of kinase inhibitors on VCIP135 S130 phosphorylation and VCIP135 DUB activity, nocodazole-arrested mitotic cells were treated with 2 μM staurosporine for 45 min or with 20 μM roscovitine or 20 μM U0126 for 2 h, respectively (Zhang )."

"We next determined the kinase responsible for VCIP135 S130 phosphorylation in mitosis. Because S130 matches the minimal S/T-P consensus motif for both Cdk1 (Nigg, 1993) and ERK1/2 (Davis, 1993; Songyang ), we applied inhibitors to these protein kinases to mitotic cells and investigated the effects on S130 phosphorylation of VCIP135. Flag-tagged VCIP135 was immunoprecipitated and analyzed by Western blotting using the pSer-130 antibody (Figure 6A). Treatment of cells with staurosporine (a general serine/threonine kinase inhibitor) or roscovitine (a Cdk inhibitor) abolished the pSer130 signal. In contrast, treatment of cells with U0126 (a MEK inhibitor that inhibits ERK1/2 activation) had no effect on S130 phosphorylation."

"As a key regulator of Golgi membrane fusion, the activity of VCIP135 is tightly controlled by phosphorylation at S130 during the cell cycle progression. We have developed a phospho-antibody that specifically recognizes phosphorylated S130 on VCIP135 (Figure 3)."

"Taken together, our results demonstrate that phosphorylation of VCIP135 on S130 in early mitosis inactivates its DUB activity for mitotic Golgi disassembly; dephosphorylation in telophase reactivates VCIP135 to promote p97/p47-mediated postmitotic Golgi membrane fusion."

"Therefore the function of VCIP135 regulated by S130 phosphorylation should be limited to postmitotic Golgi membrane fusion."

"These results demonstrate that VCIP135 S130 phosphorylation regulates its DUB activity."

"Next we determined how VCIP135 phosphorylation at S130 regulates VCIP135's deubiquitinase activity during the cell cycle."

"Our in vitro Golgi reassembly assay confirmed that VCIP135 phosphorylation at S130 attenuates p97/p47-mediated Golgi membrane fusion (Figure 7A)."

"To determine the phosphorylation state of VCIP135 on S130 during cell cycle progression, we developed an antibody that specifically recognizes phosphorylated S130 on VCIP135."

"The pSer130 antibody specifically recognizes phosphorylated S130 on VCIP135."

"To determine the phosphorylation state of VCIP135 on S130 during cell cycle progression, we developed an antibody that specifically recognizes phosphorylated S130 on VCIP135. We used the phosphopeptide KLL[pS]PILARY as the antigen, because this sequence is well conserved between species (Figure 2D). The generated antibody, here referred to as “the VCIP135 pSer130 antibody,” specifically recognizes VCIP135 immunoprecipitated from mitotic but not from interphase cells (Figure 3A, top panel), consistent with the mobility shift of VCIP135 in mitosis (Figure 3A, bottom panel). We further confirmed its specificity using VCIP135 phosphomutants immunoprecipitated from interphase (Figure 3B) or mitotic cells (Figure 3C). The VCIP135 pSer130 antibody recognized the S130E and 13E mutants in both interphase and mitotic cells, but had no immunoreactivity to the S130A and 13A mutants, regardless of the cell cycle stage (Figure 3, B and C). WT VCIP135 and the C218S, 12A, and 12E mutants were phosphorylated at S130 and were recognized by the pSer130 antibody only in mitotic cells."

"Dephosphorylation of S130 on VCIP135 upon staurosporine or roscovitine but not U0126 treatment also significantly increased the DUB activity of endogenous VCIP135 (Figure 6B), consistent with the hypothesis that phosphorylation at S130 inactivates VCIP135."

"These results demonstrate that phosphorylation of VCIP135 at S130 attenuates p97/p47-mediated Golgi membrane fusion."

"Because S130 matches the minimal S/T-P consensus motif for both Cdk1 (Nigg, 1993) and ERK1/2 (Davis, 1993; Songyang ), we applied inhibitors to these protein kinases to mitotic cells and investigated the effects on S130 phosphorylation of VCIP135."

"VCIP135 S130 is phosphorylated in prometaphase and dephosphorylated in telophase."

"Generation of an antibody that specifically recognizes phosphorylated S130 on VCIP135."

"Phosphorylation of VCIP135 on S130 attenuates p97/p47-mediated Golgi membrane fusion."

"Because the deubiquitinase activity of VCIP135 is required for p97/p47-mediated postmitotic Golgi cisternal regrowth (Wang ) and phosphorylation at S130 inhibits VCIP135 DUB activity, it is likely that cells control the DUB activity of VCIP135 by phosphorylation and thereby regulate Golgi membrane dynamics in the cell cycle."

"These results demonstrate that phosphorylation of VCIP135 at S130 controls its deubiquitinase activity in vivo. As this site is conserved between different species ranging from Xenopus to human (Figure 2D), phosphorylation of S130 may be a common mechanism for inactivation of this enzyme in mitosis."

"These results demonstrate that phosphorylation of VCIP135 at S130 controls its deubiquitinase activity in vivo."

"In addition to its S130 phosphorylation, VCIP135 is also phosphorylated at multiple sites at the C-terminus, which regulates VCIP135 membrane association and p97 interaction (Zhang )."

"Given our results, we propose that VCIP135 phosphorylation on S130 in early mitosis inhibits its activity and blocks Golgi membrane fusion, resulting in mitotic Golgi fragmentation; while VCIP135 reactivation by S130 dephosphorylation in telophase initiates p97/p47-mediated Golgi membrane fusion for postmitotic Golgi reassembly."

"Mitotic phosphorylation of VCIP135 at S130 inhibits its deubiquitinase activity."

"To determine whether Cdk1 directly phosphorylates VCIP135 on S130, we performed an in vitro phosphorylation assay with streptavidin-binding peptide (SBP)-tagged VCIP135 recombinant proteins. First, we verified that the pSer130 antibody specifically recognizes only the S130E phosphomimetic mutant but not WT VCIP135 or its phosphodeficient S130A mutant (Figure 6C). When WT VCIP135 was incubated with Cdk1, S130 was phosphorylated, as indicated by the pSer130 signal (Figure 6D, lane 2)."

"We have developed a phospho-antibody that specifically recognizes phosphorylated S130 on VCIP135 (Figure 3)."

"We have developed a phospho-antibody that specifically recognizes phosphorylated S130 on VCIP135 (Figure 3). Biochemical (Figure 4) and microscopy (Figures 4 and 5) studies showed that S130 is phosphorylated from early mitosis to anaphase, which inactivates VCIP135 DUB activity. When cells progress to telophase, S130 is dephosphorylated, VCIP135 gains activity, and the Golgi apparatus starts to reassemble. Our in vitro Golgi reassembly assay confirmed that VCIP135 phosphorylation at S130 attenuates p97/p47-mediated Golgi membrane fusion (Figure 7A). In cells, depletion of VCIP135 resulted in a defect in postmitotic Golgi reassembly. Expressing an RNAi-resistant form of WT VCIP135 rescued this defect, while expressing the inactive S130E and C218S mutants did not (Figure 7, B–D), suggesting that VCIP135 DUB activity is essential for postmitotic Golgi membrane fusion. In addition to its S130 phosphorylation, VCIP135 is also phosphorylated at multiple sites at the C-terminus, which regulates VCIP135 membrane association and p97 interaction (Zhang )."

"We next determined the kinase responsible for VCIP135 S130 phosphorylation in mitosis."

"Taken together, these results demonstrate that the pSer130 antibody specifically recognizes phosphorylated S130 of VCIP135."

"Because VCIP135 DUB activity is specifically required for postmitotic Golgi membrane fusion, we determined the effects of S130 phosphorylation of VCIP135 on postmitotic Golgi reassembly in vivo. We first depleted VCIP135 in HeLa cells by RNAi and replaced it with exogenously expressed RNAi-resistant VCIP135 by transfection. We then arrested these cells in mitosis by nocodazole treatment and analyzed postmitotic Golgi reformation by microscopy 3 h after nocodazole washout (Figure 7B). In cells that expressed WT VCIP135 or its phosphorylation mutants with deubiquitinase activity (S130A, 12A, 13A), an intact Golgi reformed in telophase. In contrast, the Golgi in cells expressing the deubiquitinase-inactive C218S or S130E mutant remained fragmented (Figure 7, C and D). These results confirmed that VCIP135 reactivation in telophase by S130 dephosphorylation plays a significant role in postmitotic Golgi membrane fusion. In addition, VCIP135 12E and 13E mutants also failed to support postmitotic Golgi reassembly (Figure 7, C and D). Our previous study showed that phosphorylation at the C-terminus (e.g., 12E) inhibits VCIP135 Golgi membrane association and p97 interaction (Zhang ). These results demonstrate that the function of VCIP135 in Golgi membrane fusion is tightly regulated by phosphorylation during the cell cycle, wherein phosphorylation at S130 and the C-terminus suppress DUB activity and membrane association, respectively, while dephosphorylation stimulates DUB activity, membrane association, and p97 interaction that are required for postmitotic Golgi reassembly."

"These results demonstrate that VCIP135 is phosphorylated in mitosis on S130 and is dephosphorylated in later mitosis."

"In this study, we found that phosphorylation of VCIP135 at a single site, S130, is necessary and sufficient to inactivate its DUB activity for Golgi disassembly in early mitosis."

"Phosphorylation of VCIP135 at S130 inhibits its deubiquitinase activity."

"VCIP135 S130 phosphorylation in the cell cycle."

"The N-terminal half of VCIP135 contains a single phosphorylation site, S130 (Zhang )."