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A bipolar clamp mechanism for activation of Jak-family protein tyrosine kinases.

Barua D, Faeder JR, Haugh JM - PLoS Comput. Biol. (2009)

Bottom Line: Building on a rule-based kinetic modeling approach that considers the concerted nature and combinatorial complexity of modular protein domain interactions, we examine these mechanisms in detail, focusing on the growth hormone (GH) receptor/Jak2/SH2-Bbeta system.The modeling results suggest that, whereas Jak2-(SH2-Bbeta)(2)-Jak2 heterotetramers are scarcely expected to affect Jak2 phosphorylation, SH2-Bbeta and dimerized receptors synergistically promote Jak2 trans-activation in the context of intracellular signaling.Analysis of the results revealed a unique mechanism whereby SH2-B and receptor dimers constitute a bipolar 'clamp' that stabilizes the active configuration of two Jak2 molecules in the same macro-complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA.

ABSTRACT
Most cell surface receptors for growth factors and cytokines dimerize in order to mediate signal transduction. For many such receptors, the Janus kinase (Jak) family of non-receptor protein tyrosine kinases are recruited in pairs and juxtaposed by dimerized receptor complexes in order to activate one another by trans-phosphorylation. An alternative mechanism for Jak trans-phosphorylation has been proposed in which the phosphorylated kinase interacts with the Src homology 2 (SH2) domain of SH2-B, a unique adaptor protein with the capacity to homo-dimerize. Building on a rule-based kinetic modeling approach that considers the concerted nature and combinatorial complexity of modular protein domain interactions, we examine these mechanisms in detail, focusing on the growth hormone (GH) receptor/Jak2/SH2-Bbeta system. The modeling results suggest that, whereas Jak2-(SH2-Bbeta)(2)-Jak2 heterotetramers are scarcely expected to affect Jak2 phosphorylation, SH2-Bbeta and dimerized receptors synergistically promote Jak2 trans-activation in the context of intracellular signaling. Analysis of the results revealed a unique mechanism whereby SH2-B and receptor dimers constitute a bipolar 'clamp' that stabilizes the active configuration of two Jak2 molecules in the same macro-complex.

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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.
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pcbi-1000364-g006: 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.

Mentions: To further evaluate the roles of the functional SH2-Bβ domains, we assessed the ability of different domain mutants to antagonize the function of wild-type SH2-Bβ in cells, i.e., to act as a dominant negative (Figure 6). The Extended Cellular Model was used with the addition of the mutant SH2-Bβ species. The SH2 domain alone competes with wild-type for Jak2 binding and is an effective inhibitor at concentrations of at least 1 µM (for nanomolar concentrations of endogenous SH2-Bβ, as expected), which is 10-fold higher than the assumed value of KD,JS (Figure 6A). Inhibition by the DD alone is through dimerization with wild-type SH2-Bβ and is somewhat less effective (Figure 6B), which might be attributed to the partial neutralization of the DD through homo-dimerization. The addition of the PH domain to either the SH2 domain (functionally equivalent to the DD-mutated SH2-B analyzed in Figure 5C and 5D) or the DD results in membrane localization of the mutant SH2-Bβ and, accordingly, more potent disruption of receptor/Jak2/SH2-Bβ macro-complexes when it is expressed in excess compared with wild-type SH2-Bβ; comparing PH-SH2 and DD-PH, the former construct shows the more robust inhibition of SH2-Bβ function (Figure 6C and 6D). The predicted efficacies of these two dominant-negatives reflect the gamut of effects, both strong and subtle, discussed previously: 1) the effect of SH2-Bβ concentration, relative to its Jak2-binding affinity, on macro-complex formation; 2) antagonism of macro-complex formation at extreme SH2-Bβ concentrations, exceeding the value of χr; and 3) the ability of phosphoinositides to enhance the effective concentration of SH2-Bβ, which facilitates macro-complex formation at low SH2-Bβ concentrations and also a modest degree of Jak2-receptor association at high SH2-Bβ concentrations that is independent of SH2-Bβ dimerization.


A bipolar clamp mechanism for activation of Jak-family protein tyrosine kinases.

Barua D, Faeder JR, Haugh JM - PLoS Comput. Biol. (2009)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2667146&req=5

pcbi-1000364-g006: 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.
Mentions: To further evaluate the roles of the functional SH2-Bβ domains, we assessed the ability of different domain mutants to antagonize the function of wild-type SH2-Bβ in cells, i.e., to act as a dominant negative (Figure 6). The Extended Cellular Model was used with the addition of the mutant SH2-Bβ species. The SH2 domain alone competes with wild-type for Jak2 binding and is an effective inhibitor at concentrations of at least 1 µM (for nanomolar concentrations of endogenous SH2-Bβ, as expected), which is 10-fold higher than the assumed value of KD,JS (Figure 6A). Inhibition by the DD alone is through dimerization with wild-type SH2-Bβ and is somewhat less effective (Figure 6B), which might be attributed to the partial neutralization of the DD through homo-dimerization. The addition of the PH domain to either the SH2 domain (functionally equivalent to the DD-mutated SH2-B analyzed in Figure 5C and 5D) or the DD results in membrane localization of the mutant SH2-Bβ and, accordingly, more potent disruption of receptor/Jak2/SH2-Bβ macro-complexes when it is expressed in excess compared with wild-type SH2-Bβ; comparing PH-SH2 and DD-PH, the former construct shows the more robust inhibition of SH2-Bβ function (Figure 6C and 6D). The predicted efficacies of these two dominant-negatives reflect the gamut of effects, both strong and subtle, discussed previously: 1) the effect of SH2-Bβ concentration, relative to its Jak2-binding affinity, on macro-complex formation; 2) antagonism of macro-complex formation at extreme SH2-Bβ concentrations, exceeding the value of χr; and 3) the ability of phosphoinositides to enhance the effective concentration of SH2-Bβ, which facilitates macro-complex formation at low SH2-Bβ concentrations and also a modest degree of Jak2-receptor association at high SH2-Bβ concentrations that is independent of SH2-Bβ dimerization.

Bottom Line: Building on a rule-based kinetic modeling approach that considers the concerted nature and combinatorial complexity of modular protein domain interactions, we examine these mechanisms in detail, focusing on the growth hormone (GH) receptor/Jak2/SH2-Bbeta system.The modeling results suggest that, whereas Jak2-(SH2-Bbeta)(2)-Jak2 heterotetramers are scarcely expected to affect Jak2 phosphorylation, SH2-Bbeta and dimerized receptors synergistically promote Jak2 trans-activation in the context of intracellular signaling.Analysis of the results revealed a unique mechanism whereby SH2-B and receptor dimers constitute a bipolar 'clamp' that stabilizes the active configuration of two Jak2 molecules in the same macro-complex.

View Article: PubMed Central - PubMed

Affiliation: Department of Chemical and Biomolecular Engineering, North Carolina State University, Raleigh, North Carolina, USA.

ABSTRACT
Most cell surface receptors for growth factors and cytokines dimerize in order to mediate signal transduction. For many such receptors, the Janus kinase (Jak) family of non-receptor protein tyrosine kinases are recruited in pairs and juxtaposed by dimerized receptor complexes in order to activate one another by trans-phosphorylation. An alternative mechanism for Jak trans-phosphorylation has been proposed in which the phosphorylated kinase interacts with the Src homology 2 (SH2) domain of SH2-B, a unique adaptor protein with the capacity to homo-dimerize. Building on a rule-based kinetic modeling approach that considers the concerted nature and combinatorial complexity of modular protein domain interactions, we examine these mechanisms in detail, focusing on the growth hormone (GH) receptor/Jak2/SH2-Bbeta system. The modeling results suggest that, whereas Jak2-(SH2-Bbeta)(2)-Jak2 heterotetramers are scarcely expected to affect Jak2 phosphorylation, SH2-Bbeta and dimerized receptors synergistically promote Jak2 trans-activation in the context of intracellular signaling. Analysis of the results revealed a unique mechanism whereby SH2-B and receptor dimers constitute a bipolar 'clamp' that stabilizes the active configuration of two Jak2 molecules in the same macro-complex.

Show MeSH
Related in: MedlinePlus