Effects of the I682F mutation on JAK2's activity, structure and stabilityby Feng Li, Wei Li, Yang Liu, Yu-Xue Tong, Ping Zhou, Lin Wang, Chong Chen, Ling-Yu Zeng, Qing-Yun Wu, Xiao-Yun Wang, Kai-Lin Xu

International Journal of Biological Macromolecules

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Year
2015
DOI
10.1016/j.ijbiomac.2015.04.063
Subject
Biochemistry / Molecular Biology / Structural Biology

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International Journal of Biological Macromolecules 79 (2015) 118–125

Contents lists available at ScienceDirect

International Journal of Biological Macromolecules j ourna l ho me pa g e: www.elsev ier .com/ locate / i jb iomac

Effects of the I682F mutation on JAK2’s activity, s

Feng Lia,1, Wei Lib,1, Yang Liuc,1, Yu-Xue Tongc, Ping Zhouc, Li

Ling-Yu Zengc, Qing-Yun Wuc,∗∗, Xiao-Yun Wangd,∗, Kai-Lin X a Department of Biology, Xuzhou Medical College, No. 209 Tongshan Road, Xuzhou 221002, People’s Republi b Laiwu Central Hospital of Xinwen Mining Industry Group, Laiwu, Shandong, People’s Republic of China c Department o oad, X d College of Life n 271 a r t i c l

Article history:

Received 26 Se

Received in re

Accepted 22 A

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Keywords:

Janus kinase 2

B-cell acute ly

Structural stab roles s. JAK ever mech 82F, I , whi ore, t truct d sta echa © 2015 Elsevier B.V. All rights reserved. 1. Introduction

Janus ki been implic receptor fa family (IL-3

IL-6R), and

R) [1]. On c on downstr activator of and differen

JAK2 mutat as the JAK2 mutations f

Our previo

Abbreviati and activator o myeloprolifera

ANS, 1-anilino intrinsic fluore ∗ Correspon ∗∗ Correspon

E-mail add (X.-Y. Wang), 1 These auth disrupted the JH1/JH2 domain interactions, activated JAK2-STAT5 http://dx.doi.o 0141-8130/© nase 2 (JAK2) is a non-receptor tyrosine kinase. It has ated in signaling by the members of type II cytokine mily (e.g., interferon receptors), the GM-CSF receptor

R, IL-5R and GM-CSF-R), the gp130 receptor family (e.g., the single chain receptor (e.g., Epo-R, Tpo-R, GH-R, PRLytokine stimulating, activated JAK2 subsequently turns eam signaling pathway such as signal transducer and transcription 5 (STAT5) and leads to the proliferation tiation of the hematopoietic cells [2]. There are many ions detected in the hematological malignancies, such

V617F mutation for MPN [3,4] and JAK2 R683S (G) or B-cell acute lymphoblastic leukemia (B-ALL) [5–8]. us studies indicated that JAK2 R683S (G) mutations ons: JAK, Janus kinase; JH, JAK homology; STAT5s, ignal transducer f transcription 5; B-ALL, B-cell acute lymphoblastic leukemia; MPDs, tive disorders; ALL, acute lymphoblastic leukemia; WT, Wild type; -8-naphthalenesulfonic acid; Emax, the emission maximum of the scence. ding author. Tel.: +86 538 8242656 8430. ding authors. Tel.: +86 516 85802382; fax: +86 516 85601527. resses: qywu82@163.com (Q.-Y. Wu), xyunwang@sdau.edu.cn lihmd@163.com (K.-L. Xu). ors contributed equally to this work. signal pathway and finally led to the development of B-ALL [9].

Thus, JAK2 might be a potential therapeutically target to design specific inhibitors for diseases caused by the deregulation of down stream signaling pathway [1,2,5–8].

JAK2 is a multidomain protein possessing seven conserved JAK homology (JH) domains 1–7 [10]. The JH1 domain is a highly conserved kinase domain. The JH2 domain which was presumed to be a pseudokinase is proven to be a kinase now [3,4]. The JH2 domain phosphorylates amino acid residues Ser523 and Tyr570 and plays vital roles in keeping JAK2 in low activity [3,4]. Previous studies indicated that domain–domain interactions were important for keeping the correct conformation of multidomain proteins [11–13]. Lindauer et al. [14] implies that the JH2 domain interacts with JH1 domain and holds the JH1 domain in a closed, inactive conformation. Our previous studies also suggested that the JAK2

C618R mutation led to JAK2 activation in the absence of ligand by disrupting the JH1/JH2 domain interactions [15]. Moreover, Bandaranayake et al. [4] also indicated that the JAK2 V617F mutation rigidified the -helix C in the N lobe of JH2 domain, which facilitated the trans-phosphorylation of the JH1 domain. Further studies revealed that domain–domain interactions also involved in the JH2 domain dimerization both in pre-dimerized cytokine receptors and after receptor rearrangement. Thus, these studies suggest that the correct conformation of JH2 domain is essential for JAK2 autoinhibition. rg/10.1016/j.ijbiomac.2015.04.063 2015 Elsevier B.V. All rights reserved.f Hematology, the Affiliated Hospital of Xuzhou Medical College, No. 99 West Huaihai R

Sciences, State Key Laboratory of Crop Biology, Shandong Agricultural University, Tai’a e i n f o ptember 2014 vised form 21 April 2015 pril 2015 e 2 May 2015 mphoblastic leukemia ility a b s t r a c t

Janus kinase 2 (JAK2) plays important migration, proliferation and apoptosi lymphoblastic leukemia (B-ALL). How unknown. In order to investigation the tions were constructed. Mutations I6 and decreased its structural stability ity and structural stability. Furtherm

I682G, I682D and I682S impaired the s state. It may be this partially unfolde provides clues in understanding the mtructure and stability n Wangc, Chong Chenc, uc,∗∗ c of China uzhou 221002, People’s Republic of China 018, Shandong, People’s Republic of China in the regulation of varieties cellular processes including cell 2 I682F genetic mutation existed in the 4–8% of B-cell acute , roles of this mutation in the development of B-ALL are still anism of the JAK2 I682F mutation led to B-ALL, series of muta682G, I682D and I682S significantly increased JAK2’s activity le the I682L mutation almost had no effect on JAK2’s activhe spectroscopic experiments implied that mutations I682F, ure of JAK2 JH2 domain, and led JAK2 to the partially unfolded te that caused JAK2 I682F constitutive activation. This study nism of the JAK2 I682F mutation caused B-ALL.

F. Li et al. / International Journal of Biological Macromolecules 79 (2015) 118–125 119

To achieve their functions, domains of the mutidomain proteins are connected sequentially by the linker in the primary structure. In some cases, the sequences of linkers do not contribute to the structural stability and activity of these proteins [16], whereas in others, linkers may [17,18]. Ho residues loc tural stabili is identified dren [5–8]. acid I682 lo domain [3,4 tions [17,18 keeping the the mechan still unclear