Ab Initio Modeling and Experimental Assessment of Janus Kinase 2 (JAK2) Kinase-Pseudokinase Complex Structure
Figure 8
Mutation of interfacial residues changed JAK2 kinase activity.
(A) JAK2 mutants showed increased basal kinase activity. Activity of HA-tagged JAK2 mutants was measured by phospho-JAK2 antibodies. Total JAK2 level was measured by anti-HA antibodies. P-JAK2: phosphorylated JAK2. V: vector alone. WT: wild-type JAK2. (B) Hyperactive JAK2 mutants showed increased STAT5 activation. Activation of STAT5 was assessed using flow cytometry with Alexa647-conjugated antibodies to phospho-STAT5. Results were normalized to wild-type JAK2. (C) Hyperactive JAK2 mutants transformed BaF3/EpoR cells into factor-independent growth. Cell growth at each indicated day was measured by MTT assay. JAK2-V617F expressing cells became saturated on Day 5. WT: wild-type JAK2. (D) Mutations in JH1 reduced basal JAK2 kinase activity. Activation of STAT5 was determined as in (B). Results were normalized to wild-type JAK2.