Diacylglycerol Signaling Research Articles

Roles for class IIA phosphatidylinositol transfer protein in neurotransmission and behavioral plasticity at the sensory neuron synapses of Caenorhabditis elegans

Growing evidence suggests that sensory neuron synapses not merely pass, but actively encode sensory information and convey it to the central nervous system. The chemosensory preferences of Caenorhabditis elegans, as manifested in the direction of chemotaxis, are reversibly regulated by prior experience at the level of sensory neurons; the attractive drive is promoted by diacylglycerol (DAG) signaling, whereas the counteracting repulsive drive requires PtdIns(3,4,5)P(3) signaling. Here we report that the two opposing drives require a class IIA phosphatidylinositol transfer protein (PITP), PITP-1, which localizes to the sensory neuron synapses. In pitp-1 mutants, attraction behavior to salt is reduced, whereas conditioned repulsion from salt is eliminated: the mutants inflexibly show weak attraction behavior to salt, irrespective of prior experience. To generate flexible behavioral outputs, attraction and repulsion, PITP-1 acts in the gustatory neuron ASER and likely regulates neurotransmission from ASER, as pitp-1 mutations do not affect the ASER Ca(2+) response to sensory stimulus. Furthermore, full attraction to salt is restored in pitp-1 mutants by expression of the phosphatidylinositol transfer domain alone, and also by mutations of a DGK gene that cause accumulation of DAG, suggesting that PITP-1 serves for DAG production via phosphatidylinositol transport and, hence, regulates synaptic transmission. In addition to gustatory behavior, olfactory behaviors and osmotic avoidance are also regulated by PITP-1 in the sensory neurons that detect each sensory stimulus. Thus, PITP-1-dependent phosphatidylinositol transport is essential for sensory neuron synapses to couple sensory inputs to effective behavioral responses.

Rac GTPase-activating Protein (Rac GAP) α1-Chimaerin Undergoes Proteasomal Degradation and Is Stabilized by Diacylglycerol Signaling in Neurons

α1-Chimaerin is a neuron-specific member of the Rho GTPase-activating protein family that selectively inactivates the small GTPase Rac. It is known to regulate the structure of dendrites and dendritic spines. We describe here that under basal conditions α1-chimaerin becomes polyubiquitinated and undergoes rapid proteasomal degradation. This degradation is partly dependent on the N-terminal region that is unique to this isoform. Mimicking diacylglycerol (DAG) signaling with a phorbol ester stabilizes endogenous α1-chimaerin against degradation and causes accumulation of the protein. The stabilization requires phorbol ester binding via the C1 domain of the protein and is independent of PKC activity. In addition, overexpression of a constitutively active Rac1 mutant is sufficient to cause an accumulation of α1-chimaerin through a phospholipase C-dependent mechanism, showing that endogenous DAG signaling can also stabilize the protein. These results suggest that signaling via DAG may regulate the abundance of α1-chimaerin under physiological conditions, providing a new model for understanding how its activity could be controlled.

DGKι regulates presynaptic release during mGluR-dependent LTD

Diacylglycerol (DAG) is an important lipid second messenger. DAG signalling is terminated by conversion of DAG to phosphatidic acid (PA) by diacylglycerol kinases (DGKs). The neuronal synapse is a major site of DAG production and action; however, how DGKs are targeted to subcellular sites of DAG generation is largely unknown. We report here that postsynaptic density (PSD)-95 family proteins interact with and promote synaptic localization of DGKι. In addition, we establish that DGKι acts presynaptically, a function that contrasts with the known postsynaptic function of DGKζ, a close relative of DGKι. Deficiency of DGKι in mice does not affect dendritic spines, but leads to a small increase in presynaptic release probability. In addition, DGKι-/- synapses show a reduction in metabotropic glutamate receptor-dependent long-term depression (mGluR-LTD) at neonatal (∼2 weeks) stages that involve suppression of a decrease in presynaptic release probability. Inhibition of protein kinase C normalizes presynaptic release probability and mGluR-LTD at DGKι-/- synapses. These results suggest that DGKι requires PSD-95 family proteins for synaptic localization and regulates presynaptic DAG signalling and neurotransmitter release during mGluR-LTD.

Melatonin Sensitizes Human Myometrial Cells to Oxytocin in a Protein Kinase Cα/Extracellular-Signal Regulated Kinase-Dependent Manner

Studies have shown that labor occurs primarily in the night/morning hours. Recently, we identified the human myometrium as a target for melatonin (MEL), the neuroendocrine output signal coding for circadian night. The purpose of this study was to determine the signaling pathway underlying the effects of MEL on contractility and the contractile machinery in immortalized human myometrial cells. To ascertain the signaling pathway of MEL leading to its effects on myometrial contractility in vitro, we performed gel retraction assays with cells exposed to iodo-MEL (I-MEL) with or without oxytocin and the Rho kinase inhibitor Y27632. I-MEL effects on inositol trisphosphate (IP(3))/diacylglycerol (DAG)/protein kinase C (PKC) signaling were also investigated. Additionally, we assayed for caldesmon phosphorylation and ERK1/2 activation. I-MEL was found to activate PKC alpha via the phospholipase C/IP(3)/DAG signaling pathway, which was confirmed by PKC enzyme assay. I-MEL did not affect myosin light chain phosphatase activity, and its effects on contractility were insensitive to Rho kinase inhibition. I-MEL did increase phosphorylation of ERK1/2 and caldesmon, which was inhibited by the MAPK kinase inhibitor PD98059 or the PKC inhibitor C1. MEL sensitizes myometrial cells to subsequent procontractile signals in vitro through activation of the phospholipase C/IP(3)/DAG signaling pathway, resulting in specific activation of PKC alpha and ERK1/2, thereby phosphorylating caldesmon, which increases actin availability for myosin binding and cross-bridging. In vivo, this sensitization would provide a mechanism for the increased nocturnal uterine contractility and labor that has been observed in late-term human pregnancy.

Diacylglycerol Kinase δ and Protein Kinase Cα Modulate Epidermal Growth Factor Receptor Abundance and Degradation through Ubiquitin-specific Protease 8

Many human epithelial cancers are characterized by abnormal activation of the epidermal growth factor receptor (EGFR), which is often caused by its excessive expression in tumor cells. The abundance of EGFR is modulated, in part, by its ubiquitination, which targets it for degradation. The components responsible for adding ubiquitin to EGFR are well characterized, but this is a reversible process, and the mechanisms that modulate the removal of ubiquitin from the EGFR are not well known. We found that de-ubiquitination of EGFR was regulated by diacylglycerol kinase delta (DGKdelta), a lipid kinase that terminates diacylglycerol signaling. In DGKdelta-deficient cells, ubiquitination of EGFR was enhanced, which attenuated the steady-state levels of EGFR and promoted its ligand-induced degradation. These effects were not caused by changes in the ubiquitinating apparatus, but instead were due to reduced expression of the de-ubiquitinase, ubiquitin-specific protease 8 (USP8). Depletion of protein kinase Calpha (PKCalpha), a target of diacylglycerol, rescued the levels of USP8 and normalized EGFR degradation in DGKdelta-deficient cells. Moreover, the effects of PKCalpha were caused by its inhibition of Akt, which stabilizes USP8. Our data indicate a novel mechanism where DGKdelta and PKCalpha modulate the levels of ubiquitinated EGFR through Akt and USP8.

Intact T cell receptor signaling by CD4+ T cells cultured in the rotating wall‐vessel bioreactor

T lymphocytes fail to proliferate or secrete cytokines in response to T cell receptor (TCR) agonists during culture in spaceflight or ground-based microgravity analogs such as rotating wall-vessel (RWV) bioreactors. In RWVs, these responses can be rescued by co-stimulation with sub-mitogenic doses of the diacyl glycerol (DAG) mimetic phorbol myristate acetate. Based on this result we hypothesized that TCR activation is abrogated in the RWV due to impaired DAG signaling downstream of the TCR. To test this hypothesis we compared TCR-induced signal transduction by primary, human, CD4(+) T cells in RWV, and static culture. Surprisingly, we found little evidence of impaired DAG signaling in the RWV. Upstream of DAG, the tyrosine phosphorylation of several key components of the TCR-proximal signal was not affected by culture in the RWV. Similarly, the phosphorylation and compartmentalization of ERK and the degradation of IkappaB were unchanged by culture in the RWV indicating that RAS- and PKC-mediated signaling downstream of DAG are also unaffected by simulated microgravity. We conclude from these data that TCR signaling through DAG remains intact during culture in the RWV, and that the loss of functional T cell activation in this venue derives from the affect of simulated microgravity on cellular processes that are independent of the canonical TCR pathway.

Negative regulation of diacylglycerol kinase θ mediates adenosine-dependent hepatocyte preconditioning

In liver ischemic preconditioning (IP), stimulation of adenosine A2a receptors (A2aR) prevents ischemia/reperfusion injury by promoting diacylglycerol-mediated activation of protein kinase C (PKC). By concerting diacylglycerol to phosphatidic acid, diacylglycerol kinases (DGKs) act as terminator of diacylglycerol signalling. This study investigates the role of DGK in the development of hepatocyte IP. DGK activity and cell viability were evaluated in isolated rat hepatocytes preconditioned by 10 min hypoxia followed by 10 min re-oxygenation or by the treatment with the A2aR agonist, CGS21680, and subsequently exposed to prolonged hypoxia. We observed that after IP or A2aR activation, a decrease in DGK activity was associated with the onset of hepatocyte tolerance to hypoxia. CGS21680-induced stimulation of A2aR specifically inhibited DGK isoform theta by activating RhoA-GTPase. Consistently, both siRNA-mediated downregulation of DGK theta and hepatocyte pretreatment with the DGK inhibitor R59949 induced cell tolerance to hypoxia. The pharmacological inhibition of DGK was associated with the diacylglycerol-dependent activation of PKC delta and epsilon and of their downstream target p38 MAPK. In conclusion, we unveil a novel signalling pathway contributing to the onset of hepatocyte preconditioning, which through RhoA-GTPase, couples A2aR to the downregulation of DGK. Such an inhibition is essential for the sustained accumulation of diacylglycerol required for triggering PKC-mediated survival signals.

Diacylglycerol kinases in the regulation of dendritic spines

Diacylglycerol (DAG) is an important lipid-signaling molecule that binds and activates various downstream effectors. Tight control over the production and removal of DAG is important in maintaining the dynamic responses of the DAG signaling system to a changing environment. Diacylglycerol kinases (DGKs) are enzymes that convert DAG to phosphatidic acid (PA). This conversion terminates DAG signaling and, at the same time, initiates additional signaling events downstream of PA, which also acts as a lipid-signaling molecule. However, little is known about how (or if) DGKs are targeted to specific subcellular sites or how DGKs tightly regulate local DAG and PA signaling. Dendritic spines are tiny protrusions on neuronal dendrites that receive the majority of excitatory synaptic inputs. They are also the sites where DAG molecules are produced through activation of postsynaptic receptors, including metabotropic glutamate receptors and NMDA receptors. Accumulating evidence indicates that synaptic levels of DAG and PA are important determinants of dendritic spine stability and that the DGKzeta isoform at excitatory postsynaptic sites is critically involved in spine maintenance. In addition, DGKzeta appears to form a multi-protein complex with functionally related proteins to organize efficient DAG and PA signaling pathways at excitatory synapses.

Diacylglycerol-Stimulated Endocytosis of Transferrin in Trypanosomatids Is Dependent on Tyrosine Kinase Activity

Small molecule regulation of cell function is an understudied area of trypanosomatid biology. In Trypanosoma brucei diacylglycerol (DAG) stimulates endocytosis of transferrin (Tf). However, it is not known whether other trypanosomatidae respond similarly to the lipid. Further, the biochemical pathways involved in DAG signaling to the endocytic system in T. brucei are unknown, as the parasite genome does not encode canonical DAG receptors (e.g. C1-domains). We established that DAG stimulates endocytosis of Tf in Leishmania major, and we evaluated possible effector enzymes in the pathway with multiple approaches. First, a heterologously expressed glycosylphosphatidylinositol phospholipase C (GPI-PLC) activated endocytosis of Tf 300% in L. major. Second, exogenous phorbol ester and DAGs promoted Tf endocytosis in L. major. In search of possible effectors of DAG signaling, we discovered a novel C1-like domain (i.e. C1_5) in trypanosomatids, and we identified protein Tyr kinases (PTKs) linked with C1_5 domains in T. brucei, T. cruzi, and L. major. Consequently, we hypothesized that trypanosome PTKs might be effector enzymes for DAG signaling. General uptake of Tf was reduced by inhibitors of either Ser/Thr or Tyr kinases. However, DAG-stimulated endocytosis of Tf was blocked only by an inhibitor of PTKs, in both T. brucei and L. major. We conclude that (i) DAG activates Tf endocytosis in L. major, and that (ii) PTKs are effectors of DAG-stimulated endocytosis of Tf in trypanosomatids. DAG-stimulated endocytosis of Tf may be a T. brucei adaptation to compete effectively with host cells for vertebrate Tf in blood, since DAG does not enhance endocytosis of Tf in human cells.

Functional links between diacylglycerol and phosphatidylinositol signaling systems in human leukocyte-derived cell lines

In leukocytes, diacylglycerol (DAG) regulates the small GTPase Ras through the agency of Ras guanyl releasing proteins (RasGRPs). Ras is thought to regulate phosphatidylinositol 3-kinase (PI3K). Therefore, DAG signaling is hypothesized to impact PI3K activity. The DAG analogue “pico” was used to activate RasGRPs in leukocyte-derived cell lines. PI3K signaling was monitored using antibodies that recognize the activated form of the PI3K-regulated protein kinase Akt. Diverse responses were documented. Some cell lines exhibit a DAG analogue-stimulated increase in phospho-Akt but this response proceeded even when Ras activation was blocked. In some Epstein–Barr virus-associated malignant cell lines and transformed B cells, high basal phospho-Akt was decreased by DAG analogue treatment. The pan-PKC inhibitor Bisindolymaleimide I blocked this effect. Basal phospho-Akt was also decreased by treatment with Go6976, an inhibitor of conventional protein kinase C and protein kinase D. While the proposed RasGRP-Ras-PI3K-Akt signaling axis may operate in some situations, our results indicate that alternative links between DAG targets such as protein kinases and the PI3K signaling system are more prominent.

A technique for monitoring multiple signals with a combination of prism-based total internal reflection fluorescence microscopy and epifluorescence microscopy

Physiological phenomena are regulated by multiple signal pathways upon receptor stimulation. Here, we have introduced a new technique with a combination of prism-based total internal reflection fluorescence microscopy (PBTIRFM) and epifluorescence microscopy (EPI) to simultaneously monitor multiple signal pathways. This instrumentation allows us to visualize three signal pathways, Ca2+, cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), and diacylglycerol (DAG)/protein kinase C (PKC) signals in living cells. Three fluorescent indicators were employed for this purpose: (1) Fura-2 AM as a calcium sensor; (2) Epac1-camp, a cyan fluorescent protein-yellow fluorescent protein fluorescence resonance energy transfer-based cAMP indicator, as a cAMP sensor; and (3) C1-tagged monomeric red fluorescent protein, a tandem DAG-binding domain of PKC gamma, as a DAG sensor or myristoylated alanine-rich C kinase substrate-tagged DsRed for the PKC activation pathway. The DAG signal was monitored by PBTIRFM, whereas the Ca2+ and cAMP signals were monitored by EPI. Adenosine trisphosphate resulted in generation of all three second messengers in triple probe-loaded Cos-7 cells. The spectral overlap between these signal probes was evaluated by means of linear unmixing. Forskolin also evoked Ca2+, cAMP/PKA, and DAG/PKC signals whereas acetylcholine activated Ca2+ and DAG/PKC signals as well as inhibiting cAMP generation in triple probe-loaded insulin-secreting cells. Thus, the optical observation system combining PBTIRFM and EPI offers a great advance in analyzing interplay of multiple signaling pathways, such as these second messengers, upon G-protein-coupled receptor stimulation in living cells.

A lipid kinase controls the maintenance of dendritic spines.

A lipid kinase controls the maintenance of dendritic spines.

Protein Kinase D Is an Essential Regulator of C. elegans Innate Immunity

Protein kinase D (PKD) mediates signal transduction downstream from phospholipase C and diacylglycerol (DAG). PKDs are activated by hormones and stress in cell lines, but little is known about PKD functions and regulation in vivo. Here, we show that DKF-2, a C. elegans PKD, regulates innate immunity. Animals lacking DKF-2 were hypersensitive to killing by bacteria that are pathogens of C. elegans and humans. DKF-2 induced 85 mRNAs, which encode antimicrobial peptides and proteins that sustain intestinal epithelium. Induction of immune effector mRNAs by DKF-2 proceeded via PMK-1 (p38 Map-kinase)-dependent and -independent pathways. TPA-1, a PKCdelta homolog, regulated activation and functions of DKF-2 in vivo. Therefore, DKF-2 provides a molecular link that couples DAG signaling to regulation of immunity. This intersection between DAG-TPA-1-DKF-2 and PMK-1 pathways enables integrated immune responses to multiple stimuli. Thus, a PKD mobilizes activation of host immune defenses against pathogens by previously unappreciated signaling pathways and mechanisms.

Lipid activation of protein kinases

Lipids acutely control the amplitude, duration, and subcellular location of signaling by lipid second messenger-responsive kinases. Typically, this activation is controlled by membrane-targeting modules that allosterically control the function of kinase domains within the same polypeptide. Protein kinase C (PKC) has served as the archetypal lipid-regulated kinase, providing a prototype for lipid-controlled kinase activation that is followed by kinases throughout the kinome, including its close cousin, Akt (protein kinase B). This review addresses the molecular mechanisms by which PKC and Akt transduce signals propagated by the two major lipid second messenger pathways in cells, those of diacylglycerol signaling and phosphatidylinositol-3,4,5-trisphosphate (PIP3) signaling, respectively.

The class II Histone deacetylase 7 controls Interleukin 2 receptor expression and proliferation of primary cytotoxic T cells (84.8)

Abstract Regulation of chromatin accessibility via class II histone deacetylases is a critical mechanism controlling gene expression. The paradigm for class II HDAC regulation is that in quiescent lymphocytes these molecules localise to the nucleus and form repressive complexes on chromatin. Antigen receptor activation of Protein Kinase D induces phosphorylation and nuclear export of HDACs thereby relieving their repressive actions on chromatin and gene expression. In this context, the present study has examined the regulation and function the class II histone deacetylase 7 in effector cytotoxic T cells (CTLs). This HDAC has been shown previously to function as a signal dependent repressor of gene transcription during T cell development in the thymus. We now show that HDAC7 is a critical regulator of Interleukin 2 receptor expression and proliferation in primary CTL. Strikingly, there is constitutive and sustained phosphorylation and export of HDAC7 from the nucleus to the cytosol in CTLs. The cytosolic localisation of HDAC7 is determined by the phosphorylation of key serine residues in the molecule S156, S322, S457 and by a balance of src kinase and diacylglycerol signalling pathways that increase its nuclear export and block its nuclear import. An HDAC7 mutant (S156A, S322A, S457A) that cannot be phosphorylated is thus maintained in the nucleus and prevents expression of the Interleukin2 (IL-2) receptor and accordingly inhibits CTL responses to IL-2. HDAC7 thus acts as a molecular switch to mediate IL-2 receptor expression and cellular responsiveness to IL-2 in primary T cells.

Synaptic removal of diacylglycerol by DGKζ and PSD-95 regulates dendritic spine maintenance

Diacylglycerol (DAG) is an important lipid signalling molecule that exerts an effect on various effector proteins including protein kinase C. A main mechanism for DAG removal is to convert it to phosphatidic acid (PA) by DAG kinases (DGKs). However, it is not well understood how DGKs are targeted to specific subcellular sites and tightly regulates DAG levels. The neuronal synapse is a prominent site of DAG production. Here, we show that DGKzeta is targeted to excitatory synapses through its direct interaction with the postsynaptic PDZ scaffold PSD-95. Overexpression of DGKzeta in cultured neurons increases the number of dendritic spines, which receive the majority of excitatory synaptic inputs, in a manner requiring its catalytic activity and PSD-95 binding. Conversely, DGKzeta knockdown reduces spine density. Mice deficient in DGKzeta expression show reduced spine density and excitatory synaptic transmission. Time-lapse imaging indicates that DGKzeta is required for spine maintenance but not formation. We propose that PSD-95 targets DGKzeta to synaptic DAG-producing receptors to tightly couple synaptic DAG production to its conversion to PA for the maintenance of spine density.

Characterization of the Differential Roles of the Twin C1a and C1b Domains of Protein Kinase Cδ

Classic and novel protein kinase C (PKC) isozymes contain two zinc finger motifs, designated "C1a" and "C1b" domains, which constitute the recognition modules for the second messenger diacylglycerol (DAG) or the phorbol esters. However, the individual contributions of these tandem C1 domains to PKC function and, reciprocally, the influence of protein context on their function remain uncertain. In the present study, we prepared PKCdelta constructs in which the individual C1a and C1b domains were deleted, swapped, or substituted for one another to explore these issues. As isolated fragments, both the deltaC1a and deltaC1b domains potently bound phorbol esters, but the binding of [(3)H]phorbol 12,13-dibutyrate ([(3)H]PDBu) by the deltaC1a domain depended much more on the presence of phosphatidylserine than did that of the deltaC1b domain. In intact PKCdelta, the deltaC1b domain played the dominant role in [(3)H]PDBu binding, membrane translocation, and down-regulation. A contribution from the deltaC1a domain was nonetheless evident, as shown by retention of [(3)H]PDBu binding at reduced affinity, by increased [(3)H]PDBu affinity upon expression of a second deltaC1a domain substituting for the deltaC1b domain, and by loss of persistent plasma membrane translocation for PKCdelta expressing only the deltaC1b domain, but its contribution was less than predicted from the activity of the isolated domain. Switching the position of the deltaC1b domain to the normal position of the deltaC1a domain (or vice versa) had no apparent effect on the response to phorbol esters, suggesting that the specific position of the C1 domain within PKCdelta was not the primary determinant of its activity.

Identification of an Autoinhibitory Mechanism That Restricts C1 Domain-mediated Activation of the Rac-GAP α2-Chimaerin

Chimaerins are a family of GTPase activating proteins (GAPs) for the small G-protein Rac that have gained recent attention due to their important roles in development, cancer, neuritogenesis, and T-cell function. Like protein kinase C isozymes, chimaerins possess a C1 domain capable of binding phorbol esters and the lipid second messenger diacylglycerol (DAG) in vitro. Here we identified an autoinhibitory mechanism in alpha2-chimaerin that restricts access of phorbol esters and DAG, thereby limiting its activation. Although phorbol 12-myristate 13-acetate (PMA) caused limited translocation of wild-type alpha2-chimaerin to the plasma membrane, deletion of either N- or C-terminal regions greatly sensitize alpha2-chimaerin for intracellular redistribution and activation. Based on modeling analysis that revealed an occlusion of the ligand binding site in the alpha2-chimaerin C1 domain, we identified key amino acids that stabilize the inactive conformation. Mutation of these sites renders alpha2-chimaerin hypersensitive to C1 ligands, as reflected by its enhanced ability to translocate in response to PMA and to inhibit Rac activity and cell migration. Notably, in contrast to PMA, epidermal growth factor promotes full translocation of alpha2-chimaerin in a phospholipase C-dependent manner, but not of a C1 domain mutant with reduced affinity for DAG (P216A-alpha2-chimaerin). Therefore, DAG generation and binding to the C1 domain are required but not sufficient for epidermal growth factor-induced alpha2-chimaerin membrane association. Our studies suggest a role for DAG in anchoring rather than activation of alpha2-chimaerin. Like other DAG/phorbol ester receptors, including protein kinase C isozymes, alpha2-chimaerin is subject to autoinhibition by intramolecular contacts, suggesting a highly regulated mechanism for the activation of this Rac-GAP.

Synergistic control of T cell development and tumor suppression by diacylglycerol kinase α and ζ

Diacylglycerol (DAG) kinases (DGKs) are a family of enzymes that convert DAG to phosphatidic acid (PA), the physiologic functions of which have been poorly defined. We report here that DGK alpha and zeta synergistically promote T cell maturation in the thymus. Absence of both DGKalpha and zeta (DGKalpha(-/-)zeta(-/-)) results in a severe decrease in the number of CD4(+)CD8(-) and CD4(-)CD8(+) single-positive thymocytes correlating with increased DAG-mediated signaling. Positive selection, but not negative selection, is impaired in DGKalpha(-/-)zeta(-/-) mice. The developmental blockage in DGKalpha(-/-)zeta(-/-) mice can be partially overcome by treatment with PA. Furthermore, decreased DGK activity also promotes thymic lymphomagenesis accompanying elevated Ras and Erk1/2 activation. Our data demonstrate a synergistic and critical role of DGK isoforms in T cell development and tumor suppression, and indicate that DGKs not only terminate DAG signaling but also initiate PA signaling in thymocytes to promote positive selection.

Wealth of opportunity - the C1 domain as a target for drug development.

The diacylglycerol-responsive C1 domains of protein kinase C and of the related classes of signaling proteins represent highly attractive targets for drug development. The signaling functions that are regulated by C1 domains are central to cellular control, thereby impacting many pathological conditions. Our understanding of the diacylglycerol signaling pathways provides great confidence in the utility of intervention in these pathways for treatment of cancer and other conditions. Multiple compounds directed at these signaling proteins, including compounds directed at the C1 domains, are currently in clinical trials, providing strong validation for these targets. Extensive understanding of the structure and function of C1 domains, coupled with detailed insights into the molecular details of ligand - C1 domain interactions, provides a solid basis for rational and semi-rational drug design. Finally, the complexity of the factors contributing to ligand - C1 domain interactions affords abundant opportunities for manipulation of selectivity; indeed, substantially selective compounds have already been identified.