Figure 1.
Molecular species and interactions considered in our models.
Three models of increasing complexity were formulated and analyzed, as described under Methods. The In Vitro Model considers the enhancement of Jak2 (J) autophosphorylation by SH2-Bβ (S) in solution and includes 11 species (dashed triangle). Two Jak2 phosphorylation sites are considered: Y1, which when phosphorylated (Y1∼P) engages SH2-Bβ, and Y2, which when phosphorylated (Y2∼P) enhances the kinase activity. The model considers the best-case scenario where Y1 is constitutively (or rapidly) phosphorylated. The Simplified Cellular Model includes GH ligand (L) and GH receptor (R) and also considers Jak2 species with Y1 dephosphorylated in the cytosol (dashed square). In this model, Jak2 binds constitutively to receptors, but binding of two Jak2 molecules in the same complex is required for Jak2 autophosphorylation. Finally, the Extended Cellular Model additionally considers phosphoinositide (P) lipids, which mediate localization of SH2-Bβ to the plasma membrane. The complex shown in the upper right depicts the ‘bipolar clamp’ mechanism whereby SH2-Bβ stabilizes macro-complexes containing two Jak2 molecules.
Figure 2.
Critical analysis of the SH2-Bβ-mediated Jak2 autophosphorylation mechanism in vitro.
(A–C) Surface and contour plots of Jak2 autophosphorylation (Y2∼P) for varying concentrations and dimerization KD values of SH2-Bβ, and with three different KD values of Jak2/SH2-Bβ binding: (A) KD,JS = 100 nM; (B) KD,JS = 10 nM; (C) KD,JS = 1 nM. See text for a description of the model assumptions, following [14]. (D) Surface and contour plot of heterotetramer (JS2J) concentration for KD,JS = 1 nM.
Figure 3.
SH2-Bβ significantly enhances GH receptor-mediated Jak2 autophosphorylation in vivo.
Steady-state calculations were performed using the Simplified Cellular Model, with equal Jak2 and SH2-Bβ concentrations and KD values (JTot = STot = KD,JS = KD,SS = 100 nM). In the SH2-B null case, STot = 0, and in the DD-mutated SH2-B case, KD,SS = infinity; these two cases are functionally equivalent (and therefore the curves lie on top of one another). (A) SH2-Bβ does not affect GH dose-dependent receptor-dimerization (all of the filled symbols in panel A lie approximately on top of one another) but mediates ∼3-fold improvement in pair-wise recruitment of Jak2 to receptors (the number of Jak2 molecules engaged in receptor-Jak2 complexes containing two Jak2). (B) Accordingly, SH2-Bβ enhances Jak2 autophosphorylation (site Y2) by roughly 3-fold.
Figure 4.
SH2-Bβ dimerization coordinates the formation of macro-complexes containing two Jak2 molecules bound to GH-dimerized receptors.
Steady-state calculations were performed using the Simplified Cellular Model and the same parameter values as in Figure 3, except with 10 nM GH stimulation and varying SH2-Bβ concentration. (A) Receptor-bound, phosphorylated Jak2 (Y2∼P), for various values of the SH2-Bβ dimerization affinity. The extreme cases of KD,SS equal to zero and infinity correspond to irreversible and no dimerization, respectively; intermediate KD,SS values are 10 nM, 100 nM, 1 µM, and 10 µM. (B) Analysis of receptor/Jak2 complexes, with KD,SS = 100 nM. SH2-Bβ dimerization coordinates the binding of two Jak2 molecules to dimerized receptors, while affecting overall receptor/Jak2 binding only modestly. Complexes containing more than two Jak2 molecules (e.g., J(RLR)JS2J) are rare.
Figure 5.
Membrane localization and dimerization of SH2-Bβ synergize to enhance the potency of its Jak2 activation-promoting function.
Steady-state calculations were performed using the Extended Cellular Model and the same parameter values as in Figure 4B. The total concentration of phosphoinositide, on a whole-cell basis, is either 0, 100 nM, or 1 µM as indicated, and its recruitment of SH2-Bβ PH domain is characterized by KD,SP = 100 nM. Two scenarios are considered: full-length SH2-Bβ (A and B) and SH2-Bβ with the dimerization domain absent (C and D). The calculated quantities are receptor-bound, phosphorylated Jak2 (Y2∼P; A and C) and total receptor-bound Jak2 (B and D).
Figure 6.
Potencies of SH2B-β domain mutants as dominant negatives antagonizing wild-type SH2-Bβ function.
Jak2 phosphorylation was calculated using the Extended Cellular Model as in Figure 5A, with the same parameter values and PTot = 1 µM. To this model, we added one of the following SH2-Bβ constructs: SH2 only (A), DD only (B), PH-SH2 (C), and DD-PH (D). As indicated, the value of the overall inhibitor concentration was either 0 (no inhibition; same as Figure 5A), 100 nM, 1 µM, or 10 µM.