A vascular gene trap screen defines RasGRP3 as an angiogenesis-regulated gene required for the endothelial response to phorbol esters

We identified Ras guanine-releasing protein 3 (RasGRP3) as a guanine exchange factor expressed in blood vessels via an embryonic stem (ES) cell-based gene trap screen to identify novel vascular genes. RasGRP3 is expressed in embryonic blood vessels, down-regulated in mature adult vessels, and reexpressed in newly formed vessels during pregnancy and tumorigenesis. This expression pattern is consistent with an angiogenic function for RasGRP3. Although a loss-of-function mutation in RasGRP3 did not affect viability, RasGRP3 was up-regulated in response to vascular endothelial growth factor (VEGF) stimulation of human umbilical vein endothelial cells, placing RasGRP3 regulation downstream of VEGF signaling. Phorbol esters mimic the second messenger diacylglycerol (DAG) in activating both protein kinase C (PKC) and non-PKC phorbol ester receptors such as RasGRP3. ES cell-derived wild-type blood vessels exposed to phorbol myristate acetate (PMA) underwent extensive aberrant morphogenesis that resulted in the formation of large endothelial sheets rather than properly branched vessels. This response to PMA was completely dependent on the presence of RasGRP3, as mutant vessels were refractory to the treatment. Taken together, these findings show that endothelial RasGRP3 is up-regulated in response to VEGF stimulation and that RasGRP3 functions as an endothelial cell phorbol ester receptor in a pathway whose stimulation perturbs normal angiogenesis. This suggests that RasGRP3 activity may exacerbate vascular complications in diseases characterized by excess DAG, such as diabetes.     

A diacylglycerol-protein kinase C-RasGRP1 pathway directs Ras activation upon antigen receptor stimulation of T cells

Ras GTPases are on/off switches regulating numerous cellular responses by signaling to various effector molecules. In T lymphocytes, Ras can be activated by two Ras exchange factors, SOS and RasGRP1, which are recruited through the adapters Grb2 and LAT and via the second-messenger diacylglycerol (DAG), respectively. Mitogen-activated protein (MAP) kinase phosphorylation patterns induced by active Ras can vary and contribute to distinct cellular responses. The different consequences of Ras activation by either guanine exchange factor are unknown. DAG also recruits and activates the kinase protein kinase Ctheta (PKCtheta) turning on the Erk MAP kinase pathway, but the biochemical mechanism responsible is unclear. We generated T-cell clones deficient in phorbol myristate acetate (a surrogate for DAG)-induced Ras activation. Analysis of a RasGRP1-deficient Jurkat T-cell clone and RasGRP1 RNA interference in wild-type cells revealed that RasGRP1 is required for optimal, antigen receptor-triggered Ras-Erk activation. RasGRP1 relies on its DAG-binding domain to selectively activate Erk kinases. Activation of Erk correlates with the phosphorylation of threonine residue 184 in RasGRP1. This phosphorylation event requires the activities of novel PKC kinases. Conversely, active PKCtheta depends on RasGRP1 sufficiency to effectively trigger downstream events. Last, DAG-PKC-RasGRP1-driven Ras-Erk activation in T cells is a unique signaling event, not simply compensated for by SOS activity.     

Mechanism of diacylglycerol-induced membrane targeting and activation of protein kinase Cdelta

The regulatory domains of novel protein kinases C (PKC) contain two C1 domains (C1A and C1B), which have been identified as the interaction site for sn-1,2-diacylglycerol (DAG) and phorbol ester, and a C2 domain that may be involved in interaction with lipids and/or proteins. Although recent reports have indicated that C1A and C1B domains of conventional PKCs play different roles in their DAG-mediated membrane binding and activation, the individual roles of C1A and C1B domains in the DAG-mediated activation of novel PKCs have not been fully understood. In this study, we determined the roles of C1A and C1B domains of PKCdelta by means of in vitro lipid binding analyses and cellular protein translocation measurements. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCdelta have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCdelta mutants showed that the C1A domain is critical for the DAG-induced membrane binding and activation of PKCdelta. The studies also indicated that an anionic residue, Glu(177), in the C1A domain plays a key role in controlling the DAG accessibility of the conformationally restricted C1A domain in a phosphatidylserine-dependent manner. Cell studies with enhanced green fluorescent protein-tagged PKCdelta and mutants showed that because of its phosphatidylserine specificity PKCdelta preferentially translocated to the plasma membrane under the conditions in which DAG is randomly distributed among intracellular membranes of HEK293 cells. Collectively, these results provide new insight into the differential roles of C1 domains in the DAG-induced membrane activation of PKCdelta and the origin of its specific subcellular localization in response to DAG.     

Beta phorbol ester- and diacylglycerol-induced augmentation of transmitter release is mediated by Munc13s and not by PKCs.

Munc13-1 is a presynaptic protein with an essential role in synaptic vesicle priming. It contains a diacylglycerol (DAG)/beta phorbol ester binding C(1) domain and is a potential target of the DAG second messenger pathway that may act in parallel with PKCs. Using genetically modified mice that express a DAG/beta phorbol ester binding-deficient Munc13-1(H567K) variant instead of the wild-type protein, we determined the relative contribution of PKCs and Munc13-1 to DAG/beta phorbol ester-dependent regulation of neurotransmitter release. We show that Munc13s are the main presynaptic DAG/beta phorbol ester receptors in hippocampal neurons. Modulation of Munc13-1 activity by second messengers via the DAG/beta phorbol ester binding C(1) domain is essential for use-dependent alterations of synaptic efficacy and survival.     

Differential membrane binding and diacylglycerol recognition by C1 domains of RasGRPs

RasGRPs (guanine-nucleotide-releasing proteins) are exchange factors for membrane-bound GTPases. All RasGRP family members contain C1 domains which, in other proteins, bind DAG (diacylglycerol) and thus mediate the proximal signal-transduction events induced by this lipid second messenger. The presence of C1 domains suggests that all RasGRPs could be regulated by membrane translocation driven by C1-DAG interactions. This has been demonstrated for RasGRP1 and RasGRP3, but has not been tested directly for RasGRP2, RasGRP4alpha and RasGRP4beta. Sequence alignments indicate that all RasGRP C1 domains have the potential to bind DAG. In cells, the isolated C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha co-localize with membranes and relocalize in response to DAG, whereas the C1 domains of RasGRP2 and RasGRP4beta do not. Only the C1 domains of RasGRP1, RasGRP3 and RasGRP4alpha recognize DAG as a ligand within phospholipid vesicles and do so with differential affinities. Other lipid second messengers were screened as ligands for RasGRP C1 domains, but none was found to serve as an alternative to DAG. All of the RasGRP C1 domains bound to vesicles which contained a high concentration of anionic phospholipids, indicating that this could provide a DAG-independent mechanism for membrane binding by C1 domains. This concept was supported by demonstrating that the C1 domain of RasGRP2 could functionally replace the membrane-binding role of the C1 domain within RasGRP1, despite the inability of the RasGRP2 C1 domain to bind DAG. The RasGRP4beta C1 domain was non-functional when inserted into either RasGRP1 or RasGRP4, implying that the alternative splicing which produces this C1 domain eliminates its contribution to membrane binding.     

Activation mechanisms of conventional protein kinase C isoforms are determined by the ligand affinity and conformational flexibility of their C1 domains

The regulatory domains of conventional and novel protein kinases C (PKC) have two C1 domains (C1A and C1B) that have been identified as the interaction site for diacylglycerol (DAG) and phorbol ester. It has been reported that C1A and C1B domains of individual PKC isoforms play different roles in their membrane binding and activation; however, DAG affinity of individual C1 domains has not been quantitatively determined. In this study, we measured the affinity of isolated C1A and C1B domains of two conventional PKCs, PKCalpha and PKCgamma, for soluble and membrane-incorporated DAG and phorbol ester by isothermal calorimetry and surface plasmon resonance. The C1A and C1B domains of PKCalpha have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. In contrast, the C1A and C1b domains of PKCgamma have comparably high affinities for both DAG and phorbol ester. Consistent with these results, mutational studies of full-length proteins showed that the C1A domain is critical for the DAG-induced activation of PKCalpha, whereas both C1A and C1B domains are involved in the DAG-induced activation of PKCgamma. Further mutational studies in conjunction with in vitro activity assay and monolayer penetration analysis indicated that, unlike the C1A domain of PKCalpha, neither the C1A nor the C1B domain of PKCgamma is conformationally restricted. Cell studies with enhanced green fluorescent protein-tagged PKCs showed that PKCalpha did not translocate to the plasma membrane in response to DAG at a basal intracellular calcium concentration due to the inaccessibility of its C1A domain, whereas PKCgamma rapidly translocated to the plasma membrane under the same conditions. These data suggest that differential activation mechanisms of PKC isoforms are determined by the DAG affinity and conformational flexibility of their C1 domains.     

New insights into the regulation of protein kinase C and novel phorbol ester receptors.

Protein kinase C (PKC), a family of related serine-threonine kinases, is a key player in the cellular responses mediated by the second messenger diacylglycerol (DAG) and the phorbol ester tumor promoters. The traditional view of PKCs as DAG/phospholipid-regulated proteins has expanded in the last few years by three seminal discoveries. First, PKC activity and maturation is controlled by autophosphorylation and transphosphorylation mechanisms, which includes phosphorylation of PKC isozymes by phosphoinositide-dependent protein kinases (PDKs) and tyrosine kinases. Second, PKC activity and localization are regulated by direct interaction with different types of interacting proteins. Protein-protein interactions are now recognized as important mechanisms that target individual PKCs to different intracellular compartments and confer selectivity by associating individual isozymes with specific substrates. Last, the discovery of novel phorbol ester receptors lacking kinase activity allows us to speculate that some of the biological responses elicited by phorbol esters or by activation of receptors coupled to elevation in DAG levels could be mediated by PKC-independent pathways.     

Regulation of RasGRP via a Phorbol Ester-Responsive C1 Domain

As part of a cDNA library screen for clones that induce transformation of NIH 3T3 fibroblasts, we have isolated a cDNA encoding the murine homolog of the guanine nucleotide exchange factor RasGRP. A point mutation predicted to prevent interaction with Ras abolished the ability of murine RasGRP (mRasGRP) to transform fibroblasts and to activate mitogen-activated protein kinases (MAP kinases). MAP kinase activation via mRasGRP was enhanced by coexpression of H-, K-, and N-Ras and was partially suppressed by coexpression of dominant negative forms of H- and K-Ras. The C terminus of mRasGRP contains a pair of EF hands and a C1 domain which is very similar to the phorbol ester- and diacylglycerol-binding C1 domains of protein kinase Cs. The EF hands could be deleted without affecting the ability of mRasGRP to transform NIH 3T3 cells. In contrast, deletion of the C1 domain or an adjacent cluster of basic amino acids eliminated the transforming activity of mRasGRP. Transformation and MAP kinase activation via mRasGRP were restored if the deleted C1 domain was replaced either by a membrane-localizing prenylation signal or by a diacylglycerol- and phorbol ester-binding C1 domain of protein kinase C. The transforming activity of mRasGRP could be regulated by phorbol ester when serum concentrations were low, and this effect of phorbol ester was dependent on the C1 domain of mRasGRP. The C1 domain could also confer phorbol myristate acetate-regulated transforming activity on a prenylation-defective mutant of K-Ras. The C1 domain mediated the translocation of mRasGRP to cell membranes in response to either phorbol ester or serum stimulation. These results suggest that the primary mechanism of activation of mRasGRP in fibroblasts is through its recruitment to diacylglycerol-enriched membranes. mRasGRP is expressed in lymphoid tissues and the brain, as well as in some lymphoid cell lines. In these cells, RasGRP has the potential to serve as a direct link between receptors which stimulate diacylglycerol-generating phospholipase Cs and the activation of Ras.     

Munc13-1-mediated vesicle priming contributes to secretory amyloid precursor protein processing

The amyloid precursor protein (APP) gives rise toc beta-amyloid peptides, which are the main constituents of senile plaques in brains of Alzheimer's disease patients. Non-amyloidogenic processing of the APP can be stimulated by phorbol esters (PEs) and by intracellular diacylglycerol (DAG) generation. This led to the hypothesis that classical and novel protein kinase Cs (PKCs), which are activated by DAG/PEs, regulate APP processing. However, in addition to PKCs, there are other DAG/PE receptors present in neurons that may participate in the modulation of APP processing. Munc13-1, a presynaptic protein with an essential role in synaptic vesicle priming, represents such an alternative target of the DAG second messenger pathway. Using Munc13-1 knock-out mice and knock-in mice expressing a Munc13-1(H567K) variant deficient in DAG/PE binding, we determined the relative contributions of PKCs and Munc13-1 to PE-stimulated secretory APP processing. We establish that, in addition to PKC, Munc13-1 significantly contributes to the regulation of secretory APP metabolism.     

Diacylglycerol kinase zeta regulates Ras activation by a novel mechanism

Guanine nucleotide exchange factors (GEFs) activate Ras by facilitating its GTP binding. Ras guanyl nucleotide-releasing protein (GRP) was recently identified as a Ras GEF that has a diacylglycerol (DAG)-binding C1 domain. Its exchange factor activity is regulated by local availability of signaling DAG. DAG kinases (DGKs) metabolize DAG by converting it to phosphatidic acid. Because they can attenuate local accumulation of signaling DAG, DGKs may regulate RasGRP activity and, consequently, activation of Ras. DGK zeta, but not other DGKs, completely eliminated Ras activation induced by RasGRP, and DGK activity was required for this mechanism. DGK zeta also coimmunoprecipitated and colocalized with RasGRP, indicating that these proteins associate in a signaling complex. Coimmunoprecipitation of DGK zeta and RasGRP was enhanced in the presence of phorbol esters, which are DAG analogues that cannot be metabolized by DGKs, suggesting that DAG signaling can induce their interaction. Finally, overexpression of kinase-dead DGK zeta in Jurkat cells prolonged Ras activation after ligation of the T cell receptor. Thus, we have identified a novel way to regulate Ras activation: through DGK zeta, which controls local accumulation of DAG that would otherwise activate RasGRP.     

Interfacial Partitioning of a Loop Hinge Residue Contributes to Diacylglycerol Affinity of Conserved Region 1 Domains

Conventional and novel isoenzymes of PKC are activated by the membrane-embedded second messenger diacylglycerol (DAG) through its interactions with the C1 regulatory domain. The affinity of C1 domains to DAG varies considerably among PKCs. To gain insight into the origin of differential DAG affinities, we conducted high-resolution NMR studies of C1B domain from PKCδ (C1Bδ) and its W252Y variant. The W252Y mutation was previously shown to render C1Bδ less responsive to DAG (Dries, D. R., Gallegos, L. L., and Newton, A. C. (2007) A single residue in the C1 domain sensitizes novel protein kinase C isoforms to cellular diacylglycerol production. J. Biol. Chem. 282, 826–830) and thereby emulate the behavior of C1B domains from conventional PKCs that have a conserved Tyr at the equivalent position. Our data revealed that W252Y mutation did not perturb the conformation of C1Bδ in solution but significantly reduced its propensity to partition into a membrane-mimicking environment in the absence of DAG. Using detergent micelles doped with a paramagnetic lipid, we determined that both the residue identity at position 252 and complexation with diacylglycerol influence the geometry of C1Bδ-micelle interactions. In addition, we identified the C-terminal helix α1 of C1Bδ as an interaction site with the head groups of phosphatidylserine, a known activator of PKCδ. Taken together, our studies (i) reveal the identities of C1Bδ residues involved in interactions with membrane-mimicking environment, DAG, and phosphatidylserine, as well as the affinities associated with each event and (ii) suggest that the initial ligand-independent membrane recruitment of C1B domains, which is greatly facilitated by the interfacial partitioning of Trp-252, is responsible, at least in part, for the differential DAG affinities.     

Live-imaging of PKC Translocation in Sf9 Cells and in Aplysia Sensory Neurons

Protein kinase Cs (PKCs) are serine threonine kinases that play a central role in regulating a wide variety of cellular processes such as cell growth and learning and memory. There are four known families of PKC isoforms in vertebrates: classical PKCs (α, βI, βII and γ), novel type I PKCs (ε and η), novel type II PKCs (δ and θ), and atypical PKCs (ζ and ι). The classical PKCs are activated by Ca²⁺ and diacylclycerol (DAG), while the novel PKCs are activated by DAG, but are Ca²⁺-independent. The atypical PKCs are activated by neither Ca²⁺ nor DAG. In Aplysia californica, our model system to study memory formation, there are three nervous system specific PKC isoforms one from each major class, namely the conventional PKC Apl I, the novel type I PKC Apl II and the atypical PKC Apl III. PKCs are lipid-activated kinases and thus activation of classical and novel PKCs in response to extracellular signals has been frequently correlated with PKC translocation from the cytoplasm to the plasma membrane. Therefore, visualizing PKC translocation in real time in live cells has become an invaluable tool for elucidating the signal transduction pathways that lead to PKC activation. For instance, this technique has allowed for us to establish that different isoforms of PKC translocate under different conditions to mediate distinct types of synaptic plasticity and that serotonin (5HT) activation of PKC Apl II requires production of both DAG and phosphatidic acid (PA) for translocation ^1-2. Importantly, the ability to visualize the same neuron repeatedly has allowed us, for example, to measure desensitization of the PKC response in exquisite detail ³. In this video, we demonstrate each step of preparing Sf9 cell cultures, cultures of Aplysia sensory neurons have been described in another video article ⁴, expressing fluorescently tagged PKCs in Sf9 cells and in Aplysia sensory neurons and live-imaging of PKC translocation in response to different activators using laser-scanning microscopy.Download video file.^{(60M, mov)}     

Diacylglycerol-dependent binding recruits PKCtheta and RasGRP1 C1 domains to specific subcellular localizations in living T lymphocytes

Diacylglycerol (DAG) signaling relies on the presence of conserved domain 1 (C1) in its target proteins. Phospholipase C-dependent generation of DAG after T cell receptor (TCR) triggering is essential for the correct immune response onset. Accordingly, two C1-containing proteins expressed in T lymphocytes, Ras guanyl nucleotide-releasing protein1 (RasGRP1) and protein kinase C (PKC), were shown to be fundamental for T-cell activation and proliferation. Although containing the same regulatory domain, they are proposed to relocate to distinct subcellular locations in response to TCR triggering. Here we studied intracellular localization of RasGRP1 and PKC C1 domains in living Jurkat T cells. The results demonstrate that, in the absence of significant primary sequence differences, the C1 domains of these proteins show specific localization within the cell and distinct responses to pharmacological stimulation and TCR triggering. These differences help explain the divergent localization and distinct functional roles of the full-length proteins, which contains them. The properties of these DAG-binding modules allow their characterization as functional markers that discriminate between DAG pools. Finally, we show that by binding to different diacylglycerol forms, overexpression of distinct C1 modules can attenuate DAG-dependent signals originating from the plasma or internal membranes. This is shown by analyzing the contribution of these two lipid pools to PLC-dependent Ras activation in response to TCR triggering.     

Diacylglycerol-induced membrane targeting and activation of protein kinase Cepsilon: mechanistic differences between protein kinases Cdelta and Cepsilon

Two novel protein kinases C (PKC), PKCdelta and PKCepsilon, have been reported to have opposing functions in some mammalian cells. To understand the basis of their distinct cellular functions and regulation, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKCepsilon and compared it with that of PKCdelta. The regulatory domains of novel PKC contain a C2 domain and a tandem repeat of C1 domains (C1A and C1B), which have been identified as the interaction site for DAG and phorbol ester. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCepsilon have comparably high affinities for DAG and phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCepsilon mutants showed that both the C1A and C1B domains play a role in the DAG-induced membrane binding and activation of PKCepsilon. The C1 domains of PKCepsilon are not conformationally restricted and readily accessible for DAG binding unlike those of PKCdelta. Consequently, phosphatidylserine-dependent unleashing of C1 domains seen with PKCdelta was not necessary for PKCepsilon. Cell studies with fluorescent protein-tagged PKCs showed that, due to the lack of lipid headgroup selectivity, PKCepsilon translocated to both the plasma membrane and the nuclear membrane, whereas PKCdelta migrates specifically to the plasma membrane under the conditions in which DAG is evenly distributed among intracellular membranes of HEK293 cells. Also, PKCepsilon translocated much faster than PKCdelta due to conformational flexibility of its C1 domains. Collectively, these results provide new insight into the differential activation mechanisms of PKCdelta and PKCepsilon based on different structural and functional properties of their C1 domains.     

RasGRP3 mediates phorbol ester-induced, protein kinase C-independent exocytosis

Phorbol esters are involved in neurotransmitter release and hormone secretion via activation of protein kinase C (PKC). In addition, it has been recently reported to enhance neurotransmitter release in a PKC-independent manner. However, the exocytotic machinery is not fully clarified. Nowadays members of the RasGRP family are being identified as novel molecules binding to diacylglycerol and calcium, representing a new class of guanine nucleotide exchange factor that activates small GTPases including Ras and Rap1. In the present study, we demonstrated that RasGRP3 is expressed in endocrine tissues and mediates phorbol ester-induced exocytosis. Furthermore, the effects were partially blocked by PKC inhibitor but not mitogen-activated protein kinase kinase inhibitor, although both significantly suppressed the phorbol ester-induced phosphorylation of extracellular signal-regulated kinase 1/2. These results indicate that RasGRP3 is implicated in phorbol ester-induced, PKC-independent exocytosis.     

Diacylglycerols containing Omega 3 and Omega 6 fatty acids bind to RasGRP and modulate MAP kinase activation

We elucidated the effects of different diacylglycerols (DAGs), i.e. 1-stearoyl-2-arachidonoyl-sn-glycerol (SAG), 1-stearoyl-2-docosahexaenoyl-sn-glycerol (SDG), and 1-stearoyl-2-eicosapentaenoyl-sn-glycerol (SEG), on [3H]PDBu binding to RasGRP. The competition studies with these DAGs on [3H]PDBu binding to RasGRP revealed different Ki values for these DAG molecular species. Furthermore, we transfected human Jurkat T cells by a plasmid containing RasGRP and assessed the implication of endogenous DAGs on activation of MAP kinases ERK1/ERK2, induced by phorbol-12-myristate-13-acetate (PMA). In control cells, GF109203X, a protein kinase C inhibitor, inhibited ERK1/ERK2 activation. However, this agent curtailed but failed to completely diminish ERK1/ERK2 phosphorylation in RasGRP-overexpressing cells, though calphostin C, a DAG binding inhibitor, suppressed the phosphorylation of MAP kinases in these cells. In cells incubated with arachidonic acid (AA), docosahexaenoic acid (DHA), and eicosapentaenoic acid (EPA), PMA induced the production of endogenous DAGs containing these fatty acids, respectively: DAG-AA, DAG-DHA, and DAG-EPA. The inhibition of production of DAG-AA and DAG-DHA significantly inhibited MAP kinase activation in RasGRP overexpressing, but not in control, cells. Our study demonstrates that three DAG molecular species bind to RasGRP, but only DAG-AA and DAG-DHA participate in the modulation of RasGRP-mediated activation of MAP kinases in Jurkat T cells.     

Mechanism of membrane redistribution of protein kinase C by its ATP-competitive inhibitors

ATP-competitive inhibitors of PKC (protein kinase C) such as the bisindolylmaleimide GF 109203X, which interact with the ATP-binding site in the PKC molecule, have also been shown to affect several redistribution events of PKC. However, the reason why these inhibitors affect the redistribution is still controversial. In the present study, using immunoblot analysis and GFP (green fluorescent protein)-tagged PKC, we showed that, at commonly used concentrations, these ATP-competitive inhibitors alone induced redistribution of DAG (diacylglycerol)-sensitive PKCalpha, PKCbetaII, PKCdelta and PKCepsilon, but not atypical PKCzeta, to the endomembrane or the plasma membrane. Studies with deletion and point mutants showed that the DAG-sensitive C1 domain of PKC was required for membrane redistribution by these inhibitors. Furthermore, membrane redistribution was prevented by the aminosteroid PLC (phospholipase C) inhibitor U-73122, although an ATP-competitive inhibitor had no significant effect on acute DAG generation. Immunoblot analysis showed that an ATP-competitive inhibitor enhanced cell-permeable DAG analogue- or phorbol-ester-induced translocation of endogenous PKC. Furthermore, these inhibitors also enhanced [3H]phorbol 12,13-dibutyrate binding to the cytosolic fractions from PKCalpha-GFP-overexpressing cells. These results clearly demonstrate that ATP-competitive inhibitors cause redistribution of DAG-sensitive PKCs to membranes containing endogenous DAG by altering the DAG sensitivity of PKC and support the idea that the inhibitors destabilize the closed conformation of PKC and make the C1 domain accessible to DAG. Most importantly, our findings provide novel insights for the interpretation of studies using ATP-competitive inhibitors, and, especially, suggest caution about the interpretation of the relationship between the redistribution and kinase activity of PKC.     

Effects on ligand interaction and membrane translocation of the positively charged arginine residues situated along the C1 domain binding cleft in the atypical protein kinase C isoforms

The C1 domain zinc finger structure is highly conserved among the protein kinase C (PKC) superfamily members. As the interaction site for the second messenger sn-1,2-diacylglycerol (DAG) and for the phorbol esters, the C1 domain has been an important target for developing selective ligands for different PKC isoforms. However, the C1 domains of the atypical PKC members are DAG/phorbol ester-insensitive. Compared with the DAG/phorbol ester-sensitive C1 domains, the rim of the binding cleft of the atypical PKC C1 domains possesses four additional positively charged arginine residues (at positions 7, 10, 11, and 20). In this study, we showed that mutation to arginines of the four corresponding sites in the C1b domain of PKCdelta abolished its high potency for phorbol 12,13-dibutyrate in vitro, with only marginal remaining activity for phorbol 12-myristate 13-acetate in vivo. We also demonstrated both in vitro and in vivo that the loss of potency to ligands was cumulative with the introduction of the arginine residues along the rim of the binding cavity rather than the consequence of loss of a single, specific residue. Computer modeling reveals that these arginine residues reduce access of ligands to the binding cleft and change the electrostatic profile of the C1 domain surface, whereas the basic structure of the binding cleft is still maintained. Finally, mutation of the four arginine residues of the atypical PKC C1 domains to the corresponding residues in the deltaC1b domain conferred response to phorbol ester. We speculate that the arginine residues of the C1 domain of atypical PKCs may provide an opportunity for the design of ligands selective for the atypical PKCs.     

RasGRP proteins--Ras-activating factors

The Ras proteins, members of small GTP-binding protein family, are regulated through the exchange of GTP/GDP nucleotide. The activity of the Ras proteins is controlled by guanine nucleotide exchange factors (GEFs) and GTP-ase activating proteins (GAPs), which activate and inactivate G proteins respectively. Beside other, well known Ras-activating GEFs, the new class of such factors was recently described. RasGRP family, known also as CalDAG-GEF, consists of four members. C1 domain, allows them to bind diacylglycerol as well as DAG-analogs like phorbol esters. Binding of the ligand leads to activation of RasGRPs and in consequence to the activation of Ras and Rap proteins by the exchange of bounded guanine nucleotides. The signal transmitted by RasGRP is terminated as a result of DAG phosphorylation catalyzed by diacylglycerol kinase (DGK). Location of RasGRP proteins on the crossing of signaling cascades and broad tissue expression pattern involve them in many events essential for the cell function. RasGRP proteins play roles in such phenomena as: T cells maturation and functioning, B cells response, platelet aggregation, mast cells activity regulation, transformation and many other. In this review, structure and function of RasGRP proteins, as well as their role in neoplastic transformation are described.