Cullin 3-Based Ubiquitin Ligases as Master Regulators of Mammalian Cell Differentiation

Abstract

Specificity of the ubiquitin proteasome system is controlled by ubiquitin E3 ligases, including their major representatives, the multisubunit cullin-RING ubiquitin (Ub) ligases (CRLs). More than 200 different CRLs are divided into seven families according to their cullin scaffolding proteins (CUL1-7) around which they are assembled. Research over two decades has revealed that different CRL families are specialized to fulfill specific cellular functions. Whereas many CUL1-based CRLs (CRL1s) ubiquitylate cell cycle regulators, CRL4 complexes often associate with chromatin to control DNA metabolism. Based on studies about differentiation programs of mesenchymal stem cells (MSCs), including myogenesis, neurogenesis, chondrogenesis, osteogenesis and adipogenesis, we propose here that CRL3 complexes evolved to fulfill a pivotal role in mammalian cell differentiation.

Keywords: BTB proteins; Cullin 3; cullin-RING-ubiquitin ligases; cytoskeleton; differentiation; mesenchymal stem cells.

Figure 1. Remodeling of CRL3^BTB complexes mediated by CAND1 and CSN

Upon substrate-induced neddylation, CRL3s are activated to ubiquitylate a specific substrate (CSN cycle). Once substrate is consumed, CSN-mediated cullin deneddylation allows binding of CAND1 (CAND1 cycle). The protein exchange factor activity of CAND1 causes displacement of the BTB protein. The released CRL3 core complex can then reassemble with another BTB protein presumably driven by cognate substrate.

Figure 2. Multilineage differentiation programs of mesenchymal stem cells (MSCs) require specific CUL3-based CRLs

Multipotent MSCs derive from numerous vascularized tissue sources, including bone marrow, adipose and skeletal muscle. MSCs function as precursors to a variety of mature mesenchymal cell types like muscle, neuron, osteoblast and adipocyte. The differentiation of MSCs occurs in different phases. For example, adipogenesis, the differentiation of adipocytes, is characterized by two phases: the determination phase and the terminal differentiation phase. In the determination phase MSCs commit to the adipocyte lineage. Preadipocytes are morphologically not yet distinguishable from their MSC precursors. During terminal differentiation, adipocytes differentiate from preadipocytes producing lipid droplets and adipocyte-specific proteins. Whereas CRL3^BACURDs are needed for neuron as well as adipocyte differentiation and CRL3^KEAP1 is necessary for adipocyte and osteoblast differentiation, most so far identified CRL3s involved in multileanage differentiation programs are specific.

Figure 3. CRL3^BTB complexes promote differentiation by targeting negative regulators of differentiation for degradation and by impinging on signaling pathways that trigger differentiation

(A) Various differentiation programs are initiated by the ubiquitylation and degradation of transcriptions factors that are targeted by the indicated CUL3-based ubiquitin ligases. Myogenic differentiation is initiated by CRL3^KLHL40 mediated degradation of DP1, neurogenic differentiation by the CRL3^BTBD6A dependent ubiquitylation of PLZF, osteoblast differentiation by CRL3^KEAP1 mediated degradation of NRF2 and of adipogenic differentiation by the CRL3^KEAP1 ubiquitylation of CHOP and its subsequent degradation by the 26S proteasome. (B) The commitment and differentiation of MSCs towards a myogenic, neurogenic, osteogenic or adipogenic cell fate depend on a variety of signaling and transcription factor pathways controlled by CUL3-based ubiquitin ligases. Indian Hedgehog (IHH) pathway is essential for bone development. It regulates gene expression necessary for chondrogenesis and osteogenesis via the GLI family of transcription factors. GLI3R is a repressor of IHH signaling, which is ubiquitylated by CRL3^SPOP and subsequently degraded by the 26S proteasome. WNT signaling plays an essential role in cell fate determination, proliferation, and differentiation. CRL3^KLHL12 mediated degradation of DSH blocks the WNT pathway inhibiting osteogenesis. Retinoic acid (RA) influences cell differentiation, proliferation and apoptosis via expression of specific target genes. The transcription of target genes is a complex process requiring RA, nuclear receptors (RARs) as well as several coregulators such as SRC-3. In response to RA, SRC-3 is degraded in a CRL3^SPOP dependent manner.

Figure 4. CRL3^BTB-dependent remodeling of the cytoskeleton and vesicle transport adaptation during differentiation

(A) RHO GTPases change between an active GTP bound state and an inactive GDP bound state, a process influenced by several signaling pathways as indicated by red arrows. Guanine-nucleotide exchange factor (GEF) triggers the release of GDP and the formation of active GTP-RHOA. GTPase activating protein (GAP) activates hydrolysis of GTP producing inactive GDP-RHOA, which is a substrate of CRL3^BACURD ubiquitylation and subsequent degradation by the 26S proteasome. RHO-associated coiled-coil kinase (ROCK) is activated by GTP-RHOA leading to an increase of myosin light chain (MLC) phosphorylation (MLC-P). This induces actomyosin-based contractility. Furthermore, ROCK directly activates LIM kinase (LIMK), which leads to filament stabilization via cofilin phosphorylation (Cofilin-P). Degradation of RHOA via CRL3^BACURD results in actin depolymerization and destabilization (red). (B) Differentiation dependent secretion of collagen by chondrocytes is mediated by CRL3^KLHL12 and its Ca²⁺-binding coregulators, PEF1 and ALG2. During differentiation, chondrocytes secrete extracellular matrix type II and type X collagen. This requires CRL3^KLHL12 dependent mono-ubiquitylation of SEC31. In this process, CRL3^KLHL12 utilizes two Ca²⁺-binding co-substrate receptors, PEF1 and ALG2. SEC1 mono-ubiquitylation triggers the formation of large COPII coats and collagen secretion. By this mechanism, Ca²⁺ from the endoplasmatic reticulum stimulates chondrocyte differentiation via CRL3^KLHL12 and its coregulators, PEF1 and ALG2.