The BRISC deubiquitinating enzyme complex limits hematopoietic stem cell expansion by regulating JAK2 K63-ubiquitination

Abstract

Hematopoietic stem cell (HSC) homeostasis is controlled by cytokine receptor–mediated Janus kinase 2 (JAK2) signaling. We previously found that JAK2 is promptly ubiquitinated upon cytokine stimulation. Whether a competing JAK2 deubiquitination activity exists is unknown. LNK is an essential adaptor protein that constrains HSC expansion through dampening thrombopoietin (TPO)–induced JAK2 signaling. We show here that a LNK-associated lysine-63 (K63)–deubiquitinating enzyme complex, Brcc36 isopeptidase complex (BRISC), attenuates HSC expansion through control of JAK2 signaling. We pinpoint a direct interaction between the LNK SH2 domain and a phosphorylated tyrosine residue in KIAA0157 (Abraxas2), a unique and defining BRISC component. Kiaa0157 deficiency in mice led to an expansion of phenotypic and functional HSCs. Endogenous JAK2 and phospho-JAK2 were rapidly K63-ubiquitinated upon TPO stimulation, and this action was augmented in cells depleted of the BRISC core components KIAA0157, MERIT40, or BRCC36. This increase in JAK2 ubiquitination after BRISC knockdown was associated with increased TPO-mediated JAK2 activation and protein levels, and increased MPL receptor presence at the cell surface. In addition, BRISC depletion promoted membrane proximal association between the MPL receptor and pJAK2/JAK2, thus enhancing activated JAK2/MPL at the cell membrane. These findings define a novel pathway by which K63-ubiquitination promotes JAK2 stability and activation in a proteasome-independent manner. Moreover, mutations in BRCC36 are found in clonal hematopoiesis in humans. This research may shed light on the mechanistic understanding of a potential role of BRCC36 in human HSCs.

Competitive sgRNA Screen Identifies p38 MAPK as a Druggable Target to Improve HSPC Engraftment

“…The candidates were chosen from the literature based on the following criteria: (1) the knockout increased the repopulating capacity of HSCs, (2) the knockout enhanced HSC frequency and self-renewal, and (3) the knockout did not cause tumorigenesis in serial transplantations. Based on these criteria, we chose 10 candidate genes, namely Lnk [ 21 , 28 , 29 ], p18Ink4c ( p18 ) [ 30 , 31 ], Mapk14 ( p38 ) [ 32 ], Egr1 [ 33 ], Ikkb [ 34 ], p190-B ( p190 ) [ 35 ], Merit40 ( M40 ) [ 36 , 37 , 38 ], Gli1 [ 39 ], c-Cbl ( Cbl ) [ 20 ], and GzmB [ 40 ]. Prior to the screen, each sgRNA of the library was independently tested in a fluorescent reporter assay for on-target cleavage activity ( Figure S1a–c ).…”

“…In contrast to p38, Lnk and Cbl are known inhibitors of Tpo and c-Kit signaling in HSPCs, which play important roles in HSC self-renewal [ 21 ]. The inhibition of Lnk and Cbl enhances the sensitivity towards Tpo and stem cell factor, the c-Kit ligand, and results in an increased frequency of HSCs [ 20 , 36 , 38 , 48 , 49 ]. As the mechanism is different between p38 and Lnk / Cbl , we hypothesize that a combination may be beneficial.…”

Adaptor protein LNK promotes anaplastic thyroid carcinoma cell growth via 14-3-3 ε/γ binding

“…The hematopoietic cells of LNK-knockout mice were highly sensitive to a series of cytokines, including IL-3, IL-7, EPO, and TPO [25]. As a result, the JAK/STAT and ERK/MAPK pathways are highly activated [9,10]. Mice with LNK deficiency exhibited hematopoietic disorders, including a triple increase in circulating white blood cell and platelet levels, the accumulation of immature B cells in bone marrow and spleen, and the expansion of hematopoietic stem cell pools with an enhanced self-renewal ability via the JAK2 pathway [19].…”

“…It contains a pleckstrin homology (PH) domain and a Src homology 2 (SH2) domain that specifically bind to phosphorylated tyrosine residues, which mediates signal transduction, and an N-terminal prolinerich region that mediates dimerization [8,9]. Studies have shown that LNK can inhibit wild-type or mutant JAK2 signal transduction through the SH2 domain and inhibit the activation of the JAK/STAT, ERK/MAPK, and PI3K/Akt/mTOR/GSK3β pathways [9][10][11][12][13]. Clinical studies have found that LNK mutations can lead to diabetes, heart disease, kidney injury, autoimmune hepatitis, acute lymphocytic leukemia, and bone marrow proliferative malignancies [6,[13][14][15][16][17][18].…”

Functional characterization of BRCC3 mutations in acute myeloid leukemia with t(8;21)(q22;q22.1)

“…G-CSF induces tyrosine phosphorylation of JAK2 through G-CSFR which leads to enhanced proliferation [45]. Of note, in a very recent study by Donaghy et al BRCC3-mediated deubiquitination of JAK2 has been implicated in limiting hematopoietic stem cell expansion and knockdown of BRCC3 was associated with an increased K63-ubiquitination and activation of JAK2 [46]. Therefore, BRCC3 inactivation enhances JAK2 signaling by two mechanisms.…”