  The inositol-phosphate messenger inositol(1,3,4,5)tetrakisphosphate (IP4) is
essential for thymocyte positive selection by regulating plasma-membrane
association of the protein tyrosine kinase Itk downstream of the T cell
receptor (TCR). IP4 can act as a soluble analog of the phosphoinositide
3-kinase (PI3K) membrane lipid product phosphatidylinositol(3,4,5)trisphosphate
(PIP3). PIP3 recruits signaling proteins such as Itk to cellular membranes by
binding to PH and other domains. In thymocytes, low-dose IP4 binding to the Itk
PH domain surprisingly promoted and high-dose IP4 inhibited PIP3 binding of Itk
PH domains. However, the mechanisms that underlie the regulation of membrane
recruitment of Itk by IP4 and PIP3 remain unclear. The distinct Itk PH domain
ability to oligomerize is consistent with a cooperative-allosteric mode of IP4
action. However, other possibilities cannot be ruled out due to difficulties in
quantitatively measuring the interactions between Itk, IP4 and PIP3, and in
generating non-oligomerizing Itk PH domain mutants. This has hindered a full
mechanistic understanding of how IP4 controls Itk function. By combining
experimentally measured kinetics of PLC{\gamma}1 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 IP4 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 PIP3 signaling.
