CUEDC2, a novel interacting partner of the SOCS1 protein, plays important roles in the leukaemogenesis of acute myeloid leukaemia.

Downregulation of suppressor of cytokine signalling-1 (SOCS1) is one of the vital reasons for JAK1-STAT3 pathway activation in acute myeloid leukaemia (AML). CUE domain-containing 2 (CUEDC2) was a novel interacting partner of SOCS1 and a positive correlation between the expression of CUEDC2 and SOCS1 was confirmed in primary AML cells and AML cell lines without SOCS1 promoter methylation. We aimed to explore roles of CUEDC2 in regulating ubiquitin-mediated degradation of SOCS1 in the leukaemogenesis of AML.According to in vitro experiments, CUEDC2 overexpression increased the level of SOCS1 protein, suppressed JAK1-STAT3 pathway activation. The suppression of this pathway inhibited AML cells' proliferation by causing G1 arrest and enhanced AML cells' sensitivity to cytarabine and idarubicin. Similarity, downregulation of CUEDC2 produced opposite results. Knockout or low expression of CUEDC2 in mouse or AML patients displayed lower overall survival and event-free survival rates, compared with these mouse and AML patients had high-CUEDC2 expression. Mechanistic studies revealed that CUEDC2 overexpression attenuated SOCS1 ubiquitination, facilitated its stabilisation by enhancing SOCS1, Elongin C and Cullin-2 (CUL2) interactions, thus inhibited JAK1-STAT3 pathway and leukaemogenesis of AML. Therefore, our novel findings indicated that CUEDC2 interacted with SOCS1 to suppress SOCS1's ubiquitin-mediated degradation, JAK1-STAT3 pathway activation and leukaemogenesis of AML.

Effects of JAK2 V556F mutation on the JAK2's activity, structural stability and the transformation of Ba/F3 cells.

Although roles of somatic JAK2 mutations in clonally myeloproliferative neoplasms (MPNs) are well established, roles of germline JAK2 mutations in the pathogenesis of MPNs remain unclear. Recently, a novel activating, germline JAK2 F556V mutation was identified and involved in the pathogenesis of MPNs, but, its pathogenesis mechanism was still unknown. In this study, homology models of JAK2 demonstrated that F556 located between two threonine residues which interacted with ATP phosphate groups by hydrogen bonds, Thr555 with the γ-phosphate and Thr557 with the ß-phosphate in the active site of JAK2's JH2 domain. Moreover, the hydrogen bond between Thr557 and Arg715 played vital roles in sustaining the structural conformation of JH2's active site and JH1-JH2 domains' interactions. When F556 was replaced by other amino acids except Trp, the hydrogen bond, JH2 domain's structural conformation and JH1-JH2 domains' interactions disrupted for changing the helix between ß2 and ß3 strands which finally caused JAK2 activation. Mechanistic and functional studies showed that JAK2 F556V mutation disrupted JAK2 JH2 domain's activity, caused JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein may provide clues to understand the pathogenesis mechanism of JAK2 F556V mutation in the MPNs.

Mutation of the conserved G66 residue in GS region decreased structural stability and activity of arginine kinase.

Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. Amino acid residues in the guanidine specificity (GS) region play important roles in the guanidine-recognition. However, little is known about roles of amino acid residue G66 in the GS region in proteins folding, activity and structural stability. In this study, a series of G66 mutations were constructed to investigate its roles in AK's structural stability and activity. Our studies revealed that mutations in this conserved site could cause pronounced loss of activity, conformational changes and structural stability. Spectroscopic experiments indicate that G66 mutations influences AK transition from the molten globule intermediate to the native state in folding process. These results provided herein may suggest that amino acid residue G66 may play a relatively important role in AK's activity and structural stability.

Roles of amino acid residues H66 and D326 in the creatine kinase activity and structural stability.

Creatine kinase (CK) is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphocreatine to ADP in vertebrates. CK contains a pair of highly conserved amino acids (H66 and D326) which might play an important role in sustaining the compact structure of CK by linking its N- and C- terminal domains; however the mechanism is still unclear. In this study, spectroscopic, structural modeling and protein folding experiments suggested that D326A, H66P and H66P/D326A mutations led to disruption of the hydrogen bond between those two amino acid residues and form the partially unfolded state which made it easier to be inactivated and unfolded under environmental stresses, and more prone to form insoluble aggregates. The formation of insoluble aggregates would decrease levels of active CKs which may provide clues in CK deficiency disease. Moreover, these results indicated that the degree of synergism had closely relationship to the conformational changes of CK. Thus, our results provided clues for understanding the mechanism of amino acid residues outside the active site in regulating substrate synergism.

Effects of the I682F mutation on JAK2's activity, structure and stability.

Janus kinase 2 (JAK2) plays important roles in the regulation of varieties cellular processes including cell migration, proliferation and apoptosis. JAK2 I682F genetic mutation existed in the 4-8% of B-cell acute lymphoblastic leukemia (B-ALL). However, roles of this mutation in the development of B-ALL are still unknown. In order to investigation the mechanism of the JAK2 I682F mutation led to B-ALL, series of mutations were constructed. Mutations I682F, I682G, I682D and I682S significantly increased JAK2's activity and decreased its structural stability, while the I682L mutation almost had no effect on JAK2's activity and structural stability. Furthermore, the spectroscopic experiments implied that mutations I682F, I682G, I682D and I682S impaired the structure of JAK2 JH2 domain, and led JAK2 to the partially unfolded state. It may be this partially unfolded state that caused JAK2 I682F constitutive activation. This study provides clues in understanding the mechanism of the JAK2 I682F mutation caused B-ALL.