HUWE1 Is a Molecular Link Controlling RAF-1 Activity Supported by the Shoc2 Scaffold

Abstract

Scaffold proteins play a critical role in controlling the activity of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. Shoc2 is a leucine-rich repeat scaffold protein that acts as a positive modulator of ERK1/2 signaling. However, the precise mechanism by which Shoc2 modulates the activity of the ERK1/2 pathway is unclear. Here we report the identification of the E3 ubiquitin ligase HUWE1 as a binding partner and regulator of Shoc2 function. HUWE1 mediates ubiquitination and, consequently, the levels of Shoc2. Additionally, we show that both Shoc2 and HUWE1 are necessary to control the levels and ubiquitination of the Shoc2 signaling partner, RAF-1. Depletion of HUWE1 abolishes RAF-1 ubiquitination, with corresponding changes in ERK1/2 pathway activity occurring. Our results indicate that the HUWE1-mediated ubiquitination of Shoc2 is the switch that regulates the transition from an active to an inactive state of the RAF-1 kinase. Taken together, our results demonstrate that HUWE1 is a novel player involved in regulating ERK1/2 signal transmission through the Shoc2 scaffold complex.

The ubiquitin ligase Huwe1 regulates the maintenance and lymphoid commitment of hematopoietic stem cells

“…We tested several other known Huwe1 substrates, including p53, Miz1 and Mcl1 17 , 30 , 31 , but no increase in these proteins was observed. Since Huwe1 has also been implicated in MAP kinase and Wnt signaling 32 , 33 , we also measured phosphorylated Erk1/2 and beta-catenin, yet neither were affected by Huwe1 depletion. To address whether the accumulation of N-myc was proteasome-dependent, the Huwe1 knockdown study was repeated in the presence of a proteasome inhibitor.…”

Shoc2-tranduced ERK1/2 motility signals — Novel insights from functional genomics

“…Loss of Shoc2 in mammalian cultured cells and C. elegans leads to a dramatic decrease in ERK1/2 activity [17, 22, 23]. As a scaffold protein, Shoc2 provides a molecular platform for multi-protein assemblies that modulate ERK1/2 activity [24, 25]. In addition to its signaling partners Ras and RAF-1, Shoc2 tethers the catalytic subunit of protein phosphatase 1c (PP1c) as well as proteins of the ubiquitin machinery HUWE1 and PSMC5 [23, 26, 27].…”

“…In addition to its signaling partners Ras and RAF-1, Shoc2 tethers the catalytic subunit of protein phosphatase 1c (PP1c) as well as proteins of the ubiquitin machinery HUWE1 and PSMC5 [23, 26, 27]. The ability of this non-catalytic scaffold to mediate ERK1/2 signaling is controlled through allosteric ubiquitination [24]. Alterations in the mechanisms controlling ubiquitination of the scaffold affect Shoc2-mediated ERK1/2 signals and cell motility [27].…”

The FBXW7-SHOC2-Raptor Axis Controls the Cross-Talks between the RAS-ERK and mTORC1 Signaling Pathways

“…Its mammalian homolog was subsequently identified to bind to RAS/RAF by its N-terminal domain to activate MAPK cascade (Li et al, 2000). However, it is previously unknown how the turn-over of SHOC2 is regulated, although HUWE1 E3 ligase was reported to ubiquitylate SHOC2, which is not for SHOC2 degradation but rather for facilitating RAF ubiquitylation and degradation (Jang et al, 2014). We reported here that upon activation of the MAPK signal by growth factor EGF or serum, SHOC2 is phosphorylated at the Thr 507 residue within the FBXW7 consensus binding motif, which facilitates its binding to FBXW7 for subsequent ubiquitylation and degradation (Figure 2).…”

“…In pancreatic cancer cells with RAS mutations, SHOC2 knock down inhibits mitogen-activated protein kinase (MAPK) but not phosphatidylinositol 3-kinase (PI3K) activity (Rodriguez-Viciana et al, 2006), which was also seen in other types of cancer cells with active Ras (Jang et al, 2015). HUWE1 E3 ligase was reported to ubiquitylate SHOC2, not for its degradation, but for facilitating RAF ubiquitylation and degradation (Jang et al, 2014). …”