Scripps Research Logo

The Sauer Lab

Research

2.2. A novel function for soluble IP4 in positively regulating PH domain recruitment to membrane phospholipids in vivo.

One of the most interesting mouse mutants identified in a forward genetic screen in collaboration with Mike Cooke at GNF is Ms. T-less1. This mutant has no peripheral T cells due to a complete block of T cell development at the DP stage with completely impaired positive selection2, 3. We found that this developmental block is the result of a deficiency in Inositol(1,4,5)trisphosphate 3-kinase B (ItpkB), an enzyme that phosphorylates the soluble second messenger molecule Inositol(1,4,5)trisphosphate (IP3) into a higher order Inositolpolyphosphate, Inositol(1,3,4,5)tetrakisphosphate (IP4)1, 3, 4. Another group found a similar phenotype in conventional ItpkB deficient mice5. TCR induction of IP4 production in an ItpkB-dependent manner5-8 suggests that ItpkB is involved in TCR signaling, possibly through generation of IP4.

itpkB structure

Fig. 2. Schematic ItpkB Structure. ItpkB is composed of an ill characterized N-terminal presumable targeting and localization domain, followed by Ca2+/Calmodulin (CaM) or IP3 binding and catalytic domains. A point mutation abrogates ItpkB expression through introduction of a premature STOP codon at position 199 of the amino acid sequence in Ms. T-less.

A major effort in the lab is now to determine the precise mechanism(s) through which ItpkB regulates positive selection. To elucidate these processes, we combine genetic, molecular biological and biochemical analyses.

These studies led us to the exciting finding that soluble IP4 has a previously unknown in vivo function in positively regulating the function of an important signaling domain present in many proteins, the Pleckstrin Homology domain (PH domain)3. PH domains act as docking modules which allow cytosolic proteins to be recruited to cell membranes in response to cell surface receptor stimulation. PH domains do this by binding to membrane lipids such as PIP3, which are generated after receptor stimulation. It is commonly thought that PH domain recruitment is regulated by generation and turnover of their membrane ligands. Our results indicate that PH domain regulation can be much more intricate. In DP thymocytes, the TCR induced binding of several PH domains to membrane PIP3 is augmented by IP4, which can itself act as a soluble PH domain ligand. Among other proteins, this is in particular required for TCR induced membrane recruitment and activation of the Tec family protein tyrosine kinase Itk, an important mediator of TCR signaling. Thus, IP4 acts as a PH domain regulator in vivo, and this involves an unexpected, positive role. This intriguing result changes our view of how PH domain function is regulated in vivo. Finding out how many PH domains are regulated, or "helped" by IP4 is an exciting area of current research in our lab.

IP4 looks just like the PH domain binding portion of PIP3. To explain how IP4 can augment PH domain binding to PIP3, we propose two models drawing on well established mechanisms of enzyme action, the induced fit model and the cooperative allosteric model, which would require an ability of PH domains to form aggregates (Fig. 3). Interestingly, we found that PH domains can indeed form aggregates - another previously unknown aspect of PH domain function that makes the cooperative allosteric model very attractive. Finding out whether aggregation is a common feature of PH domains and how it affects their functions in vivo is another area of intense current research in the lab.

proposed model

Fig. 3. Proposed mechanistic models for IP4 augmentation of PIP3 binding to PH domains3. (A) In the "induced fit" model, IP4 binds to a PH domain and changes its conformation towards one with high affinity for IP4 and PIP3. Once the IP4-bound PH domain encounters a local excess of PIP3, for example in the cell membrane after receptor stimulation, the PIP3 simply replaces the IP4 and remains firmly bound. This way, IP4 has facilitated PH domain binding to PIP3 by inducing a better fitting conformation in the PH domain. (B) In the "cooperative allosteric" model, the PH domain pre-exists as a dimer or higher order aggregate. IP4 binding to one subunit induces conformational changes in the other subunit(s) such that they can bind PIP3 more easily and with higher affinity. As a result, even small amounts of IP4 augment binding of the aggregate PH domain to PIP3, similar to how trace amounts of a catalytic reagent accelerate chemical reactions.

Further Reading

  1. Wen, B.G. et al. Inositol (1,4,5) trisphosphate 3 kinase B controls positive selection of T cells and modulates Erk activity. Proc Natl Acad Sci U S A 101, 5604-5609 (2004).
  2. Wen, B.G. et al. (eds.) Ms. T-less, an ENU-Induced Mouse Mutant Linking Impaired Expression of Ins(1,4,5)P3 3-kinase B (Itpkb) to Defective Erk Activation and Blocked Positive Selection. (Medimont S.r.l., Bologna, Italy; 2004).
  3. Huang, Y.H. et al. Positive Regulation of Itk PH Domain Function by Soluble IP4. Science 316, 886-889. (2007).
  4. Miller, A.T. et al. Production of Ins(1,3,4,5)P4 mediated by the kinase Itpkb inhibits store-operated calcium channels and regulates B cell selection and activation. Nat Immunol 8, 514-521 (2007).
  5. Pouillon, V. et al. Inositol 1,3,4,5-tetrakisphosphate is essential for T lymphocyte development. Nat Immunol 4, 1136-1143 (2003).
  6. Guse, A.H. & Emmrich, F. T-cell receptor-mediated metabolism of inositol polyphosphates in Jurkat T-lymphocytes. Identification of a D-myo-inositol 1,2,3,4,6-pentakisphosphate-2-phosphomonoesterase activity, a D-myo-inositol 1,3,4,5,6-pentakisphosphate-1/3-phosphatase activity and a D/L-myo-inositol 1,2,4,5,6-pentakisphosphate-1/3-kinase activity. J Biol Chem 266, 24498-24502 (1991).
  7. Guse, A.H., Greiner, E., Emmrich, F. & Brand, K. Mass changes of inositol 1,3,4,5,6-pentakisphosphate and inositol hexakisphosphate during cell cycle progression in rat thymocytes. J Biol Chem 268, 7129-7133 (1993).
  8. Guse, A.H. & Emmrich, F. Determination of inositol polyphosphates from human T-lymphocyte cell lines by anion-exchange high-performance liquid chromatography and post-column derivatization. J Chromatogr 593, 157-163 (1992).
  9. Klein, D.E., Lee, A., Frank, D.W., Marks, M.S. & Lemmon, M.A. The pleckstrin homology domains of dynamin isoforms require oligomerization for high affinity phosphoinositide binding. The Journal of biological chemistry 273, 27725-27733 (1998).