Shown are the major known features of the three ITPK-gene family inositol(1,4,5)trisphosphate (InsP₃) 3-kinases, but not to scale. ITPKs contain a carboxy-terminal InsP₃-kinase domain preceded by a Ca²⁺–Calmodulin (CaM)-binding domain. Ca²⁺–CaM binding can activate ITPKA and ITPKB and is likely required for ITPKB function in thymocytes17, 85. The physiological roles of the highly divergent amino-terminal regions of the ITPKs are unknown, but they might include the regulation of ITPK activity, subcellular targeting or adaptor functions90. ITPKs have several motifs that have been implicated in actin binding, nucleo-cytoplasmic shuttling (nuclear export sequence, NES), proteolytic processing (potential PEST motifs) or phosphorylation by protein kinase A or C (PKA, PKC), or by CaMKII. Biochemical studies indicate that PKA and PKC may control ITPK activity, possibly by interfering with Ca²⁺–CaM binding. However, the in vivo relevance of all of these interactions is unknown. Several excellent reviews discuss them in detail11–15, 33, 35, 85. Modified/Reproduced with permission from REF.33.

Schematic depiction of B cell receptor (BCR) signalling through ITPKB. Following BCR engagement and assembly of a “signalosome” multiprotein-complex, phospholipases Cγ1 and -2 (PLCγ) hydrolyse phosphatidylinositol(4,5)bisphosphate (PtdInsP₂) into the second messengers diacylglycerol (DAG) and Ins(1,4,5)P₃ (InsP₃). As in T cells, InsP₃ mediates the release of Ca²⁺ from the ER by binding to InsP₃-receptors (IP₃R). The resulting depletion of Ca²⁺ stores activates plasma-membrane channels that mediate store-operated Ca²⁺ entry (SOCE). The resulting increase in intracellular Ca²⁺ levels is required for B cell proliferation and differentiation. Ca²⁺ also activates ITPKB (Fig. 3), which converts InsP₃ to Ins(1,3,4,5)P₄ (InsP₄). InsP₄ inhibits SOCE through an unknown mechanism16, 19, but can also affect InsP₃-receptors, InsP₃-dephosphorylation by 5-phosphatases, other InsP₄-effectors, or be converted to other InsPs which might mediate aspects of InsP₄-function in B cells. In any case, ITPKB acts as a feedback inhibitor of BCR-induced SOCE. Physiologically, this is required to prevent B cell anergy and extend the functional B cell repertoire by decreasing the number of B cells that are negatively selected.

Hematopoiesis originates from hematopoietic stem cells (HSC). Multipotent progenitors (MPP) give rise to common lymphoid (CLP) or myeloid (CMP) progenitors. CLPs initiate the B/T cell lineages through early-thymic-progenitors (ETP) and pro-B cells, respectively. ETPs develop through CD4^–CD8^– (DN) and CD4⁺CD8⁺ (DP) stages into mature T cells. In the bone-marrow, pro-B cells develop via pre-B cells into immature B cells. These translocate into the spleen to mature through transitional stages into mature B cells. CMPs give rise to granulocyte–monocyte (GMP) and megakaryocyte–erythrocyte (MEP) progenitors and the indicated mature cell types. In mice, ItpkB-deficiency impairs DP thymocyte positive selection, blocking maturation into CD4⁺ and CD8⁺ T cells (red bar)17, 18, 23. Human ITPKC loss-of-function may cause peripheral T cell-hyperactivation42. In ItpkB^−/− mice, perturbed splenic B cell development yields reduced numbers of B cells that are anergic16, 19, 21. Conversely, ItpkB-deficiency causes GMP-accumulation (green arrows), the generation of hyperactive neutrophilic granulocytes with decreased viability, and mildly reduced erythrocyte numbers20, 22, 47. Mast cells express ItpkB and produce IP₄ after stimulation115. Small-molecule ITPK-inhibition might augment their activation24, 109, but the target-selectivity of the low-affinity ITPK-inhibitors used is unknown and genetic studies are needed.

After antigen-receptor engagement, the membrane-lipid PtdIns(4,5)P₂ — which has a membrane-embedded lipid, and a cytosol-exposed charged inositol phosphate (InsP) moiety — can either be converted into PtdIns(3,4,5)P₃/PtdInsP₃ by phosphoinositide 3-kinase (PI3K), or into the second messengers diacylglycerol (DAG) and soluble Ins(1,4,5)P₃/InsP₃ by phospholipase-Cγ1 (PLCγ1). The phosphatase-and-tensin-homologue phosphatase PTEN counteracts PI3K. SH2 domain-containing-inositol-polyphosphate 5-phosphatases 1/2 (SHIP) convert PtdInsP₃ into PtdIns(3,4)P₂. Primarily via their InsP-headgroups, PtdIns(4,5)P_2, PtdInsP₃ and PtdIns(3,4)P₂ can bind to pleckstrin homology (PH) domains in certain proteins and recruit them to membranes. InsP₃ mobilizes Ca²⁺ but can also be phosphorylated at its 3-position into Ins(1,3,4,5)P₄/InsP₄ by InsP₃ 3-kinases (ITPKA, ITPKB, ITPKC, IPMK/IPK2)11, 17, 18, 23, 34, 35. Owing to its similarity to the PtdInsP₃-headgroup, InsP₄ can bind to certain PtdInsP₃-binding PH domains and promote (green) or inhibit (red) PtdInsP₃-binding. In CD4⁺CD8⁺ thymocytes, InsP₄-promotion of PtdInsP₃-binding to the ITK/TEC PH domains establishes a feedback-loop of PLCγ1-activation. In neutrophils, InsP₄-inhibition of PtdInsP₃- or PtdIns(3,4)P₂-binding to its PH domain may limit AKT membrane-recruitment. InsP₄ can also inhibit RASA3/GAP1^IP4BP-binding to PtdIns(4,5)P₂ or PtdInsP₃18, 21. Whether this occurs in immunocytes is unknown. R₁, R₂, fatty-acid side-chains; Circled P, phosphate moiety. Orange, enzymes with demonstrated physiological relevance in immunocytes.

TCR-engagement activates protein-tyrosine-kinases (SFK, ZAP), which tyrosine-phosphorylate transmembrane-adaptors (TMAPs, LAT). This causes phosphoinositide 3-kinase/PI3K-activation. PI3K phosphorylates membrane-phosphatidylinositol(4,5)bisphosphate/PtdIns(4,5)P₂/PIP₂ to phosphatidylinositol(3,4,5)trisphosphate/PtdInsP₃/PIP₃. By binding to their PH domains, PtdInsP₃ recruits phospholipase-Cγ1 (PLCγ1) and its upstream-activator-kinase ITK. ITK is oligomeric with low PH domain-affinity for PtdInsP₃ (dark gray). Therefore, initial ITK-recruitment is limited and only triggers low-level PLCγ1-activation. PLCγ1 hydrolyzes PtdInsP₂ into low diacylglycerol/DAG- and Ins(1,4,5)P₃/InsP₃-amounts. InsP₃ mobilizes Ca²⁺. Ca²⁺ binds calmodulin/CaM which binds to and activates ITPKs and calcineurin/CaN139. CaN dephosphorylates and activates the transcription-factor NFAT29. ITPKs phosphorylate InsP₃ into Ins(1,3,4,5)P₄/InsP₄ which may have several functions based on data from ItpkB^−/− mouse thymocytes17, 18, 23, ITPKC-mutant human T cells42 and biochemical studies11–13, 25, 51, 83, 85. InsP₄-binding to one ITK-subunit allosterically increases the PH domain affinity of all ITK-subunits for PtdInsP₃ (light gray). This promotes ITK membrane-recruitment, causing full PLCγ1-activation and sufficient DAG-production to activate RAS–ERK and trigger thymocyte positive selection. ITPKB-deficiency perturbs this feedback-activation through decreased InsP₄-production and ITK-recruitment. ITPKC-deficiency might cause peripheral T cell-hyperactivity, possibly through increased NFAT signalling. The cause could be impaired InsP₃-turnover by ITPKC or InsP₄-inhibited 5-phosphatases, or perturbed other functions of InsP₄ or its derivatives (Box 1)45, 51.