FIGURE 8.

Three βc cytoplasmic lysines are necessary for βc ubiquitination, JAK2 association, and tyrosine phosphorylation. A, stably transfected HEK293 cells expressing the indicated βc KtoR mutants in pCMV-Script were left unstimulated (0 h) or stimulated with 10 ng/ml of IL-5 for 1 h. Lysates were prepared using RIPA buffer, IP with anti-βc mAbs and analyzed by IB with the indicated antibodies (n = 3). Note the correlation of Ub-deficient βc receptors with reduced JAK2 association and low βc Tyr phosphorylation. B, same as in A, except stably transfected HEK293 cells expressing βc ΔBox 1 mutant receptors were used for the IP/IB assays. Note how βc ΔBox 1 mutants have reduced, but not absent ubiquitination even though JAK2 or JAK1 are not associated with these receptors. C, quantification of the relative amount of JAK2 co-IP with βc following IL-5 stimulation was determined by measuring JAK2 band densities from IP/IBs in A and normalized against corresponding βc band densities (n = 3). Relative associations for each βc receptor are as follows: WT βc = 0.30 ± 0.06; βc (K1–3R) = 0.09 ± 0.04; βc K(4–6R) = 0.33 ± 0.1; βc K16R 0.05 ± 0.05; βc ΔBox 1 = 0.025 ± 0.016.