In Silico Modeling of Itk Activation Kinetics in Thymocytes Suggests Competing Positive and Negative IP(4) Mediated Feedbacks Increase Robustness

The inositol-phosphate messenger inositol(1,3,4,5)tetrakisphosphate (IP(4)) is essential for thymocyte positive selection by regulating plasma-membrane association of the protein tyrosine kinase Itk downstream of the T cell receptor (TCR). IP(4) can act as a soluble analog of the phosphoinositide 3-kinase (PI3K) membrane lipid product phosphatidylinositol(3,4,5)trisphosphate (PIP(3)). PIP(3) recruits signaling proteins such as Itk to cellular membranes by binding to PH and other domains. In thymocytes, low-dose IP(4) binding to the Itk PH domain surprisingly promoted and high-dose IP(4) inhibited PIP(3) binding of Itk PH domains. However, the mechanisms that underlie the regulation of membrane recruitment of Itk by IP(4) and PIP(3) remain unclear. The distinct Itk PH domain ability to oligomerize is consistent with a cooperative-allosteric mode of IP(4) action. However, other possibilities cannot be ruled out due to difficulties in quantitatively measuring the interactions between Itk, IP(4) and PIP(3), and in generating non-oligomerizing Itk PH domain mutants. This has hindered a full mechanistic understanding of how IP(4) controls Itk function. By combining experimentally measured kinetics of PLCγ1 phosphorylation by Itk with in silico modeling of multiple Itk signaling circuits and a maximum entropy (MaxEnt) based computational approach, we show that those in silico models which are most robust against variations of protein and lipid expression levels and kinetic rates at the single cell level share a cooperative-allosteric mode of Itk regulation by IP(4) involving oligomeric Itk PH domains at the plasma membrane. This identifies MaxEnt as an excellent tool for quantifying robustness for complex TCR signaling circuits and provides testable predictions to further elucidate a controversial mechanism of PIP(3) signaling.