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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 β-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 β2 and β3 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. [ABSTRACT FROM AUTHOR]

Janus tyrosine kinase 2 (JAK2) mediates downstream signaling of cytokine receptors in all hematological lineages, constitutively active somatic JAK2 mutations play key roles in the pathology of myeloproliferative neoplasms (MPNs). Recently, germline JAK2 mutations are also associated with triple-negative MPNs. A novel germline mutation JAK2 V625F is reported to be involved in a subset of MPNs patients. However, the pathogenesis of this mutation caused MPN is still unclear. In this study, the homology models of JAK2 V625F showed that the newly formed interaction between F625 and Y613 disrupted the JAK2 JH1-JH2 domain interactions was responsible for its activation, when F625 and Y613 interaction was disrupted, its activity significantly decreased. While, when this interaction was repaired whether by forming hydrogen bond or salt bond, it would cause JAK2 activation. Biochemical studies also demonstrated that JAK2 V625F mutation led to JAK2-STAT5 pathway activation and promoted the proliferation of BaF3 cells. Thus, our results herein provide clues to understand the mechanism JAK2 V625F mutation caused MPNs and give information for the development of JAK2 mutation specific inhibitors. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Janus kinase 2 (JAK2) is an important mediator of cytokine receptor signaling and plays key roles in hematopoietic and immune responses. The acquired JAK2 R683G(S) somatic mutations are detected in 15% of patients with B-cell acute lymphoblastic leukemia (B-ALL) and are presumed to be a biomarker for B-ALL. However, how JAK2 R683G(S) mutations lead to B-ALL is still unclear. Our results indicated that the E627 and R683 interaction played a vital role in JAK2 autoinhibition. Mutations (R683S, R683G and E627A) disrupting this interaction led to JAK2 constitutive activation, while mutations (R683K, E627D) restoring this interaction decreased its activity. Furthermore, spectroscopy experiments implied that disruption of the E627 and R683 interaction abolished JH1/JH2 domain interactions and forced the JH1 domain into the open, active conformation. Mutations abolishing this interaction promoted the proliferation of Ba/F3 cells. The results herein may provide clues to understanding the mechanism of JAK2 R683G(S) mutation-associated B-ALL. [ABSTRACT FROM AUTHOR]

Abstract: Creatine kinase (CK) is a key enzyme for cellular energy metabolism, catalyzing the reversible phosphoryl transfer from phosphocreatine to ADP in vertebrates. Due to its important physiological functions, the acquired CK somatic mutations are closely correlated to diseases. In this study, the E79G point mutation was identified in two acute myocardial infarction patients with muscle CK activity deficiency. The crystal structure of CK indicates that the E79 and K138 interaction plays key roles in sustaining the recognition between N-terminal and C-terminal domains. Mutations of these residues caused pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Moreover, spectroscopic spectra experiments suggested that mutations disrupting this hydrogen bond impaired the secondary and tertiary structure of CK. Meanwhile, protein folding experiments implied that mutations lead them to the partially unfolded state which made them easier to be inactivated and unfolded under environmental stresses. Furthermore, this partially unfolded state upon environmental stresses might gradually decrease the CK level in the patients. Thus, these results might provide clues in the mechanism of CK deficiency diseases. [Copyright &y& Elsevier]

Abstract: Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. Domain–domain interactions are important to the function of multidomain proteins. However, little is known about the role of the linker in the proteins folding, activity and structural stability. In this research, a series mutation of conserved residue L113 located in the linker was mutated to explore its roles in the activity and structural stability of AK. The mutations L113D and L113K led to pronounced loss of activity and structural stability. Furthermore, spectroscopic experiments indicated that the mutations L113D and L113K impaired the structure of AK, which resulted in a partially unfolded state with more hydrophobic exposure and exposed Trp residues. The inability to fold to the functional compact state made the mutant is prone to aggregate under environmental stresses. While the mutation L113I almost had no effect on AK activity and structural stability. These results suggested that the residue L113 played important roles in sustaining the N- and C-terminal domains interactions. [Copyright &y& Elsevier]

Abstract: Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. Domain–domain interactions may be very important to the structure and functions of many multidomain proteins. However, little is known about the role of amino acid residues located in the linker between the N- and C-terminal domains in the structural stability and functions of multidomain proteins. In this research, A series mutation of conserved residue Ile121 located in the linker were mutated to explore its roles in the activity and structural stability of AK. The mutations I121D and I121K led to pronounced loss of activity and structural stability. Furthermore, these mutations also led to serious aggregation during heat-and GdnHCl-induced denaturation and refolding from the GdnHCl-denatured state. More importantly, all the mutantions except I121L could not successfully recover their activities by dilution-initiated refolding, and showed significant decreased rate constant during AK refolding. While the mutation I121L almost had no effect on AK activity and structural stability. These results suggested that mutations of the residue I121 in the linker might affect the correct positioning of the domains and thus disrupt the efficient recognition and interactions between the N- and C-terminal domains. [Copyright &y& Elsevier]

Abstract: Arginine kinase (AK) catalyzes the reversible phosphorylation of arginine by ATP, yielding the phosphoarginine. In this research, six conserved residues located on the intra-subunit domain–domain interfaces were mutated to explore their roles in the activity and structural stability of dimer AK. The mutations D69A, E70A, E71A and F80A led to pronounced loss of AK activity and structural stability. Although the mutations V75A and F76A had little effect on AK activity and structure, they caused gradually decreased the stability and reactivation of dimer AK. Our results suggested that the mutations might affect the correct positioning of the N-loop and C-loop thus disrupted the efficient recognition and interactions between the N-terminal domain and C-terminal domain which may influence the compact dimer structure, and result in decreased activity and structural stability. [Copyright &y& Elsevier]

Abstract: Arginine kinase, a member of phosphagen kinase, is a key enzyme in the cellular energy metabolism of invertebrates. A series mutation of conserved amino acid residue V65 was constructed to investigate its role in AK substrate synergism, structural stability and activity. Our study revealed that mutation in this conserved site could cause pronounced loss of activity, conformational changes and distinct substrate synergism alteration. Spectroscopic experiments indicated that these mutations influenced transition from the molten globule intermediate to the native state in folding process. These results provided herein suggest that amino acid residue V65 played a relatively important role in AK substrate synergism, structural stability and activity. [Copyright &y& Elsevier]

Risk-stratification of acute myeloid leukemia (AML) based on (cyto)genetic aberrations, including hotspot mutations, deletions and point mutations have evolved substantially in recent years. With the development of next-generation sequence technology, more and more novel mutations in the AML were identified. Thus, to unravel roles and mechanism of novel mutations would improve prognostic and predictive abilities. In this study, two novel germline JAK2 His608Tyr (H608Y) and His608Asn (H608N) mutations were identified and the molecular basis of these mutations in the leukemiagenesis of AML was elucidated. Our results indicated that JAK2 H608Y and H608N mutations disrupted the hydrogen bond between Q656 and H608 which reduced the JH2 domain's activity and abolished interactions between JH1 and JH2 domains, forced JAK2 into the active conformation, facilitated the entrance of substrates and thus caused JAK2 hyperactivation. Further studies suggested that JAK2 H608Y and H608N mutations enhanced the cell proliferation and inhibited the differentiation of Ba/F3 and MV4-11 cells via activating the JAK2-STAT5 signaling pathway. Moreover, rescue experiments demonstrated that mutations repaired the hydrogen bond between Q656 and H608 displayed opposite results. Thus, this study revealed the molecular basis of JAK2 H608Y and H608N mutations in the pathology of AML. • Two novel JAK2 H608Y and H608N germline mutations were identified in 6.5 % of AML by NGS. • JAK2 H608Y and H608N mutations abolished the interaction between Q656 and H608 led to JAK2 hyperactivation. • H608Y and H608N mutations reduced JH2 domain's activity and forced JAK2 into the open, active state. • JAK2 H608Y and H608N mutations maintained cell proliferation of Ba/F3 cells without IL-3. • JAK2 H608Y and H608N mutations promoted the pathology of AML via activating JAK2-STAT5 signaling pathway. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Highlights: [•] JAK2 R683S (G) mutations are presumed to be a biomarker for MPDs. [•] R683 located in the linker between the N and C lobes of JH2 domain which was important for JAK2 auto-inhibition. [•] Mutations R683G, R683S and R683E increased JAK2 activity and led them into partially unfolded state. [•] This study provides clues in understanding the mechanism of JAK2 R683S (G) mutations caused B-ALL. [Copyright &y& Elsevier]