Computational Models of Tandem Src Homology 2 Domain Interactions and Application to Phosphoinositide 3-Kinase

Abstract

Intracellular signal transduction proteins typically utilize multiple interaction domains for proper targeting, and thus a broad diversity of distinct signaling complexes may be assembled. Considering the coordination of only two such domains, as in tandem Src homology 2 (SH2) domain constructs, gives rise to a kinetic scheme that is not adequately described by simple models used routinely to interpret in vitro binding measurements. To analyze the interactions between tandem SH2 domains and bisphosphorylated peptides, we formulated detailed kinetic models and applied them to the phosphoinositide 3-kinase p85 regulatory subunit/platelet-derived growth factor ␤-receptor system. Data for this system from different in vitro assay platforms, including surface plasmon resonance, competition binding, and isothermal titration calorimetry, were reconciled to estimate the magnitude of the cooperativity characterizing the sequential binding of the high and low affinity SH2 domains (C-SH2 and N-SH2, respectively). Compared with values based on an effective volume approximation, the estimated cooperativity is 3 orders of magnitude lower, indicative of significant structural constraints. Homodimerization of fulllength p85 was found to be an alternative mechanism for high avidity binding to phosphorylated platelet-derived growth factor receptors, which would render the N-SH2 domain dispensable for receptor binding.Intracellular signal transduction networks, under the control of activated cell surface receptors, govern cell functional behaviors such as proliferation, migration, differentiation, and programmed cell death (1). Proper communication between signaling proteins is generally contingent upon noncovalent, intermolecular interactions, mediated by well conserved protein domains. A key feature of these domains is their modular nature, which has facilitated the extensive characterization of their binding affinities and specificities in vitro, as well as the construction of "synthetic" signaling proteins with prescribed function (2). The prototypical and best characterized interaction domains in signaling are the Src homology 2 (SH2) 3 domains, which direct interactions of proteins with receptor tyrosine kinases and other tyrosine-phosphorylated proteins (3). Receptors of the receptor tyrosine kinase family, which engage growth factor ligands such as platelet-derived growth factor (PDGF), are activated through ligand binding, receptor oligomerization, and autophosphorylation on multiple intracellular residues, which then serve as a scaffold for recruitment of proteins containing SH2 and analogous domains (4, 5).Signaling proteins typically contain three or more modular interaction domains of various types, and therefore the diversity of interactions that might take place in the cell is staggering (6). Further complicating the problem is the avidity effect, which tends to promote the cooperative association of different domains with binding partners in the same multimolecular complex or subcellular compartment. Other mechanis...

Biophysical assay for tethered signaling reactions reveals tether-controlled activity for the phosphatase SHP-1

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Systems Model of T Cell Receptor Proximal Signaling Reveals Emergent Ultrasensitivity

“…The system of ODEs representing the biochemical reaction network based on the above scheme was generated in BioNetGen [16] (see [38], [45]–[47] for additional examples of the application of BioNetGen to model reaction networks) and integrated in Matlab (Mathworks, MA). The BioNetGen file used to generate this network is available in Text S1 and contains the set of default parameters (10 reaction rates and 4 chemical concentrations).…”

Guidelines for visualizing and annotating rule-based models

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