Dataset Information

Regulation of c-Raf Stability through the CTLH Complex.

ABSTRACT: c-Raf is a central component of the extracellular signal-regulated kinase (ERK) pathway which is implicated in the development of many cancer types. RanBPM (Ran-Binding Protein M) was previously shown to inhibit c-Raf expression, but how this is achieved remains unclear. RanBPM is part of a recently identified E3 ubiquitin ligase complex, the CTLH (C-terminal to LisH) complex. Here, we show that the CTLH complex regulates c-Raf expression through a control of its degradation. Several domains of RanBPM were found necessary to regulate c-Raf levels, but only the C-terminal CRA (CT11-RanBPM) domain showed direct interaction with c-Raf. c-Raf ubiquitination and degradation is promoted by the CTLH complex. Furthermore, A-Raf and B-Raf protein levels are also regulated by the CTLH complex, indicating a common regulation of Raf family members. Finally, depletion of CTLH subunits RMND5A (required for meiotic nuclear division 5A) and RanBPM resulted in enhanced proliferation and loss of RanBPM promoted tumour growth in a mouse model. This study uncovers a new mode of control of c-Raf expression through regulation of its degradation by the CTLH complex. These findings also uncover a novel target of the CTLH complex, and suggest that the CTLH complex has activities that suppress cell transformation and tumour formation.

SUBMITTER: McTavish CJ

PROVIDER: S-EPMC6412545 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Regulation of c-Raf Stability through the CTLH Complex.

McTavish Christina J CJ Bérubé-Janzen Wesley W Wang Xu X Maitland Matthew E R MER Salemi Louisa M LM Hess David A DA Schild-Poulter Caroline C

International journal of molecular sciences 20190221 4

c-Raf is a central component of the extracellular signal-regulated kinase (ERK) pathway which is implicated in the development of many cancer types. RanBPM (Ran-Binding Protein M) was previously shown to inhibit c-Raf expression, but how this is achieved remains unclear. RanBPM is part of a recently identified E3 ubiquitin ligase complex, the CTLH (C-terminal to LisH) complex. Here, we show that the CTLH complex regulates c-Raf expression through a control of its degradation. Several domains of ...[more]

PMID: 30795516

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Project description:BackgroundHistone deacetylase 6 (HDAC6) is a microtubule-associated deacetylase that promotes many cellular processes that lead to cell transformation and tumour development. We previously documented an interaction between Ran-Binding Protein M (RanBPM) and HDAC6 and found that RanBPM expression inhibits HDAC6 activity. RanBPM is part of a putative E3 ubiquitin ligase complex, termed the C-terminal to LisH (CTLH) complex. Here, we investigated the involvement of the CTLH complex on HDAC6 inhibition and assessed the outcome of this regulation on the cellular motility induced by HDAC6.MethodsCell lines (Hela, HEK293 and immortalized mouse embryonic fibroblasts) stably or transiently downregulated for several components of the CTLH complex were employed for the assays used in this study. Interactions of HDAC6, RanBPM and muskelin were assessed by co-immunoprecipitations. Quantifications of western blot analyses were employed to evaluate acetylated ?-tubulin levels. Confocal microscopy analyses were used to determine microtubule association of HDAC6 and CTLH complex members. Cell migration was evaluated using wound healing assays.ResultsWe demonstrate that RanBPM-mediated inhibition of HDAC6 is dependent on its association with HDAC6. We show that, while HDAC6 does not require RanBPM to associate with microtubules, RanBPM association with microtubules requires HDAC6. Additionally, we show that Twa1 (Two-hybrid-associated protein 1 with RanBPM) and MAEA (Macrophage Erythroblast Attacher), two CTLH complex members, also associate with ?-tubulin and that muskelin, another component of the CTLH complex, is able to associate with HDAC6. Downregulation of CTLH complex members muskelin and Rmnd5A (Required for meiotic nuclear division homolog A) resulted in decreased acetylation of HDAC6 substrate ?-tubulin. Finally, we demonstrate that the increased cell migration resulting from downregulation of RanBPM is due to the relief in inhibition of HDAC6 ?-tubulin deacetylase activity.ConclusionsOur work shows that RanBPM, together with the CTLH complex, associates with HDAC6 and restricts cell migration through inhibition of HDAC6 activity. This study uncovers a novel function for the CTLH complex and suggests that it could have a tumour suppressive role in restricting HDAC6 oncogenic properties.

Project description:The Ras-Raf-MEK-extracellular signal-regulated kinase (ERK) pathway participates in the control of many fundamental cellular processes including proliferation, survival, and differentiation. The pathway is deregulated in up to 30% of human cancers, often due to mutations in Ras and the B-Raf isoform. Raf-1 and B-Raf can form heterodimers, and this may be important for cellular transformation. Here, we have analyzed the biochemical and biological properties of Raf-1/B-Raf heterodimers. Isolated Raf-1/B-Raf heterodimers possessed a highly increased kinase activity compared to the respective homodimers or monomers. Heterodimers between wild-type Raf-1 and B-Raf mutants with low or no kinase activity still displayed elevated kinase activity, as did heterodimers between wild-type B-Raf and kinase-negative Raf-1. In contrast, heterodimers containing both kinase-negative Raf-1 and kinase-negative B-Raf were completely inactive, suggesting that the kinase activity of the heterodimer specifically originates from Raf and that either kinase-competent Raf isoform is sufficient to confer high catalytic activity to the heterodimer. In cell lines, Raf-1/B-Raf heterodimers were found at low levels. Heterodimerization was enhanced by 14-3-3 proteins and by mitogens independently of ERK. However, ERK-induced phosphorylation of B-Raf on T753 promoted the disassembly of Raf heterodimers, and the mutation of T753 prolonged growth factor-induced heterodimerization. The B-Raf T753A mutant enhanced differentiation of PC12 cells, which was previously shown to be dependent on sustained ERK signaling. Fine mapping of the interaction sites by peptide arrays suggested a complex mode of interaction involving multiple contact sites with a main Raf-1 binding site in B-Raf encompassing T753. In summary, our data suggest that Raf-1/B-Raf heterodimerization occurs as part of the physiological activation process and that the heterodimer has distinct biochemical properties that may be important for the regulation of some biological processes.

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Regulation of c-Raf Stability through the CTLH Complex.

Publications

Regulation of c-Raf Stability through the CTLH Complex.

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