A JAK2 interdomain linker relays Epo receptor engagement signals to kinase activation.
JAK2 (Janus kinase 2) is essential for cytokine receptor signaling, and several lines of evidence support a causal role of an activating JAK2 mutation in myeloproliferative disorders. JAK2 activity is autoinhibited by its pseudokinase domain in the basal state, and the inhibition is released by cytokine stimulation; how engagement of the cognate receptor triggers this release is unknown. From a functional screen for gain-of-function JAK2 mutations, we discovered 13 missense mutations, nine in the pseudokinase domain and four in the Src homology 2 (SH2)-pseudokinase domain linker. These mutations identified determinants for autoinhibition and inducible activation in JAK2. Two of the mutants, K539I and N622I, resulted in erythrocytosis in mice. Scanning mutagenesis of the SH2-pseudokinase domain linker indicated that its N-terminal part was essential for interaction of JAK2 with the Epo receptor, whereas certain mutations in the C-terminal region conferred constitutive activation. We further showed that substitutions for Glu(543)-Asp(544) in this linker or Leu(611), Arg(683), or Phe(694) in the hinge proximal region of the pseudokinase domain resulted in activated JAK2 mutants that could not be further stimulated by Epo. These results suggest that the SH2-pseudokinase domain linker acts as a switch that relays cytokine engagement to JAK2 activation by flexing the pseudokinase domain hinge.
Zhao
Lequn
L
Department of Cell Biology, University of Texas SouthwesternMedical Center, Dallas, Texas 75390, USA.
Dong
Hongyun
H
Zhang
Cheng Cheng
CC
Kinch
Lisa
L
Osawa
Mitsujiro
M
Iacovino
Michelina
M
Grishin
Nikolai V
NV
Kyba
Michael
M
Huang
Lily Jun-shen
LJ
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D001854:Bone Marrow Cells / Q000378:metabolism; D016026:Bone Marrow Transplantation / Q000379:methods; D002460:Cell Line; D004789:Enzyme Activation; D006801:Humans; D015151:Immunoblotting; D053614:Janus Kinase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D010766:Phosphorylation; D011086:Polycythemia / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D017467:Receptors, Erythropoietin / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction; D014162:Transfection; D018909:src Homology Domains
D050505:Mutant Proteins; D017467:Receptors, Erythropoietin; D053614:Janus Kinase 2