Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao

doi:10.1016/j.bbrc.2013.02.090

Cited by 21 publications

(18 citation statements)

References 19 publications

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“…3C,D), suggesting that they contain phosphorylation sites important for regulation of membrane dissociation. This is consistent with previous phosphoproteomic studies which showed that the VCIP135 Cterminus (aa741-end) contains most of the phosphorylation sites (Dephoure et al, 2008;Olsen et al, 2010;Totsukawa et al, 2013). Therefore, we propose that phosphorylation in the C-terminus of VCIP315 inhibits its membrane association during mitosis.…”

Section: P97 Depletion Increases Vcip135 Protein Level and Membrane Asupporting

confidence: 93%

“…Recently, Totsukawa and co-workers reported that phosphorylation of VCIP135 inhibited its binding to p97 and p97-mediated Golgi assembly in vitro, and identified T760 and S767 in the C-terminus of VCIP135 as cdc2 phosphorylation sites (Totsukawa et al, 2013), but missed other sites described here, especially the important N-terminus phosphorylation site S130 that also contributes to the regulation of the VCIP135-p97 interaction (Fig. 5).…”

Section: Discussionmentioning

confidence: 57%

“…Mass spectrometry studies (Dephoure et al, 2008;Olsen et al, 2010) identified a number of sites on VCIP135 that are phosphorylated in mitosis; a recent report by Totsukawa and co-workers also showed that VCIP135 is phosphorylated by cdc2 during mitosis (Totsukawa et al, 2013). Therefore, we determined whether phosphorylation plays a role in the regulation of VCIP135 membrane association.…”

Section: P97 Depletion Increases Vcip135 Protein Level and Membrane Amentioning

confidence: 90%

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Phosphorylation regulates VCIP135 function in Golgi membrane fusion during the cell cycle

Zhang

Wang

2013

Journal of Cell Science

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The Golgi apparatus in mammalian cells consists of stacks that are often laterally linked into a ribbon-like structure. During cell division, the Golgi disassembles into tubulovesicular structures in the early stages of mitosis and reforms in the two daughter cells by the end of mitosis. Valosin-containing protein p97-p47 complex-interacting protein, p135 (VCIP135), an essential factor involved in p97-mediated membrane fusion pathways, is required for postmitotic Golgi cisternae regrowth and Golgi structure maintenance in interphase. However, how VCIP135 function is regulated in the cell cycle remains unclear. Here, we report that VCIP135 depletion by RNA interference results in Golgi fragmentation. VCIP135 function requires membrane association and p97 interaction, both of which are inhibited in mitosis by VCIP135 phosphorylation. We found that wild-type VCIP135, but not its phosphomimetic mutants, rescues Golgi structure in VCIP135-depleted cells. Our results demonstrate that VCIP135 phosphorylation regulates its Golgi membrane association and p97 interaction, and thus contributes to the tight control of the Golgi disassembly and reassembly process during the cell cycle.

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Section: P97 Depletion Increases Vcip135 Protein Level and Membrane Asupporting

confidence: 93%

Section: Discussionmentioning

confidence: 57%

Section: P97 Depletion Increases Vcip135 Protein Level and Membrane Amentioning

confidence: 90%

See 1 more Smart Citation

Phosphorylation regulates VCIP135 function in Golgi membrane fusion during the cell cycle

Zhang

Wang

2013

Journal of Cell Science

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“…Phosphorylation of Vcpip1 at pT760 by CDK1 inhibits Golgi membrane fusion, a process which is reported to be a prerequisite for entry into mitosis. We find this site to have reduced phosphorylation after PDE inhibition [38, 39]. …”

Section: Resultsmentioning

confidence: 99%

Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics

Golkowski

Shimizu‐Albergine

Suh

et al. 2016

Cellular Signalling

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Many cellular processes are modulated by cyclic AMP and nucleotide phosphodiesterases (PDEs) regulate this second messenger by catalyzing its breakdown. The major unique function of testicular Leydig cells is to produce testosterone in response to luteinizing hormone (LH). Treatment of Leydig cells with PDE inhibitors increase cAMP levels and the activity of its downstream effector, cAMP-dependent protein kinase (PKA), leading to a series of kinase-dependent signaling and transcription events that ultimately increase testosterone release. We have recently shown that PDE4B, PDE8A and PDE8B are highly expressed in rodent Leydig and adrenocortical cells and that combined inhibition of PDE4 and PDE8 lead to dramatically increased steroid biosynthesis. Here we investigated and the effect of PDE4 and PDE8 inhibitions on the molecular mechanisms of cAMP in a mouse MA10 Leydig cell line model with SILAC mass spectrometry-based phosphoproteomics. We treated MA10 cells either with PDE family specific PDE4 inhibitor (Rolipram) and PDE8 family specific inhibitor (PF-04957325) alone or in combination and quantified the resulting phosphorylation changes at five different time points between 0 and 180 minutes. We identified 28,336 phosphosites from 4837 proteins and observed significant regulation of 749 sites in response to PDE4 and PDE8 inhibitor treatment. Of these, 132 phosphosites were consensus PKA sites. Our data strongly suggest that PDE4 and PDE8 inhibitors synergistically regulate phosphorylation of proteins required for many different cellular processes, including cell cycle progression, lipid and glucose metabolism, transcription, endocytosis and vesicle transport. Our data suggests cAMP, PDE4 and PDE8 coordinate regulation of steroidogenesis by acting on not one rate-limiting step but rather multiple pathways. Moreover, the pools of cAMP controlled by these PDEs also coordinate many other metabolic processes that must be regulated to assure timely and sufficient testosterone secretion in response to LH.

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“…Both ATPases utilize specific cofactors, with SNAPs and p115 promoting NSF-mediate fusion, and complexes of p97 with p47 and VCIP135, or with p37 controlling p97 function in Golgi reformation (Kondo et al, 1997; Uchiyama et al, 2006). NSF-mediated fusion is proposed to produce larger vesicles and tubular–reticular elements, which are then fused by a p97-mediatedprocesstogeneratecisternae.Inmitosis,p47,p37,andVCIP135arephosphorylated by Cdk1, which blocks p97-mediated membrane fusion so that Golgi membranes remain disassembled (Kaneko, Tamura, Totsukawa, & Kondo, 2010; Totsukawa, Matsuo, Kubota, Taguchi, & Kondo, 2013; Uchiyama et al, 2003). Thus, mitotic phosphorylation of the membrane fusion machinery can explain the mitotic Golgi phenotype of dispersed tubular–reticular membranes and vesicles in the cytosol (Fig.…”

Section: 3 Mechanism Of Postmitotic Golgi Reassemblymentioning

confidence: 99%

Signaling at the Golgi During Mitosis

Colanzi

Sütterlin

2013

Methods for Analysis of Golgi Complex Function

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The Golgi complex of mammalian cells is composed of interconnected stacks of flattened cisternae that form a continuous membrane system in the pericentriolar region of the cell. At the onset of mitosis, this so-called Golgi ribbon is converted into small tubular–vesicular clusters in a tightly regulated fragmentation process, which leads to a temporary loss of the physical Golgi–centrosome proximity. Mitotic Golgi breakdown is required for Golgi partitioning into the two daughter cells, cell cycle progression and may contribute to the dispersal of Golgi-associated signaling molecules. Here, we review our current understanding of the mechanisms that control mitotic Golgi reorganization, its biological significance, and assays that are used to study this process.

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Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

Cited by 21 publications

References 19 publications

Phosphorylation regulates VCIP135 function in Golgi membrane fusion during the cell cycle

Phosphorylation regulates VCIP135 function in Golgi membrane fusion during the cell cycle

Studying mechanisms of cAMP and cyclic nucleotide phosphodiesterase signaling in Leydig cell function with phosphoproteomics

Signaling at the Golgi During Mitosis

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