Atypical C1 Domains Research Articles

Subcellular localization of Rap1 GTPase activator CalDAG‐GEFI is orchestrated by interaction of its atypical C1 domain with membrane phosphoinositides

BackgroundThe small GTPase Rap1 and its guanine nucleotide exchange factor, CalDAG‐GEFI (CDGI), are critical for platelet function and hemostatic plug formation. CDGI function is regulated by a calcium binding EF hand regulatory domain and an atypical C1 domain with unknown function. ObjectiveHere, we investigated whether the C1 domain controls CDGI subcellular localization, both in vitro and in vivo. MethodsCDGI interaction with phosphoinositides was studied by lipid co‐sedimentation assays and molecular dynamics simulations. Cellular localization of CDGI was studied in heterologous cells by immunofluorescence and subcellular fractionation assays. ResultsLipid co‐sedimentation studies demonstrated that the CDGI C1 domain associates with membranes through exclusive recognition of phosphoinositides, phosphatidylinositol (4,5)‐biphosphate (PIP2) and phosphatidylinositol (3,4,5)‐triphosphate (PIP3). Molecular dynamics simulations identified a phospholipid recognition motif consisting of residues exclusive to the CDGI C1 domain. Mutation of those residues abolished co‐sedimentation of the C1 domain with lipid vesicles and impaired membrane localization of CDGI in heterologous cells. ConclusionOur studies identify a novel interaction between an atypical C1 domain and phosphatidylinositol (4,5)‐biphosphate and phosphatidylinositol (3,4,5)‐triphosphate in cellular membranes, which is critical for Rap1 signaling in health and disease.

Reprogramming fatty acyl specificity of lipid kinases via C1 domain engineering.

C1 domains are lipid-binding modules that regulate membrane activation of kinases, nucleotide exchange factors, and other C1-containing proteins to trigger signal transduction. Despite annotation of typical C1 domains as diacylglycerol (DAG) and phorbol ester sensors, the function of atypical counterparts remains ill-defined. Here, we assign a key role for atypical C1 domains in mediating DAG fatty acyl specificity of diacylglycerol kinases (DGKs) in live cells. Activity-based proteomics mapped C1 probe binding as a principal differentiator of type 1 DGK active sites that combined with global metabolomics revealed a role for C1s in lipid substrate recognition. Protein engineering by C1 domain swapping demonstrated that exchange of typical and atypical C1s is functionally tolerated and can directly program DAG fatty acyl specificity of type 1 DGKs. Collectively, we describe a protein engineering strategy for studying metabolic specificity of lipid kinases to assign a role for atypical C1 domains in cell metabolism.

The C1 domain of Vav3, a novel potential therapeutic target

Vav1/2/3 comprise a protein family with guanyl nucleotide exchange activity for Rho and Rac as well as with motifs conferring adapter activity. Biologically, Vav1 plays a critical role in hematologic cell signaling, whereas Vav2/3 have a wider tissue distribution, but all 3 Vav proteins are implicated in cancer development. A structural feature of Vav1/2/3 is the presence of an atypical C1 domain, which possesses close structural homology to the typical C1 domains of protein kinase C but which fails to bind the second messenger diacylglycerol or the potent analogs, the phorbol esters. Previously, we have shown that five residues in the Vav1 C1 domain are responsible for its lack of phorbol ester binding. Here, we show that the lack of phorbol ester binding of Vav3 has a similar basis. We then explore the consequences of phorbol ester binding to a modified Vav3 in which the C1 domain has been altered to allow phorbol ester binding. We find both disruption of the guanyl nucleotide exchange activity of the modified Vav 3 as well as a shift in localization to the membrane upon phorbol ester treatment. This change in localization is associated with altered interactions with other signaling proteins. The studies provide a first step in assessing the potential for the design of custom C1 domain targeted molecules selective for the atypical C1 domains of Vav family proteins.

Abstract 5344: Understanding the structural requirements of C1 domains for phorbol ester binding, analysis of the atypical C1 domain of STAC2

Abstract C1 domains are the recognition motif for the second messenger diacylglycerol and for the phorbol esters. They play a critical role in the regulation of multiple families of signaling proteins such as protein kinase C, RasGRP, and the chimaerins. Atypical C1 domains preserve substantial homology with the diacylglycerol / phorbol ester responsive C1 domains but fail to bind these ligands. A recent advance in the field has been the recognition that the atypical C1 domains represent a continuum of structures ranging from those with measurable but weak phorbol ester binding affinity through those that preserve the phorbol ester binding cleft but have residues interfering with the insertion of the C1 domain into membranes and finally those that lack appropriate binding cleft geometry. STAC2 (SH3 and cysteine-rich domain 2) protein attracted our interest because, while its C1 domain has been classified as atypical, it had actually not been tested for phorbol ester binding activity and possesses marked homology to C1 domains that bind phorbol esters. The strategy for analysis has been to probe the influence of individual residues in the STAC2 C1 domain that are either not present or are unusual in the numerous phorbol ester responsive C1 domains. Reciprocal approaches have been to start either with the STAC2 C1 domain, mutate the individual residues, and look for gain of phorbol ester binding activity or to start with the PKC delta C1b domain and look for residues that cause reduction in binding affinity. The former approach is efficient if a single residue is responsible for the lack of binding activity; the latter approach is preferable if multiple residues are responsible. First, we confirmed that indeed the C1 domain of STAC2 is atypical and fails to bind phorbol ester. Second, we identified the residues in positions 11, 13, 24 25, 41 and 45 of the C1 domain of STAC2 as potential candidates for interfering with binding activity. However, replacement of none of these residues with the corresponding residues from PKC delta C1b sufficed to generate phorbol ester binding activity. One the other hand, introducing mutations into the C1b domain of PCK delta identified L24N, that caused a significant (~20-fold) decrease in the binding affinity. The probable basis for the negative effect of L24N on binding is that Leucine 24, together with Phenylalanine 13, Leucine 20 and Tryptophan 22, are all exposed at the top of the binding cleft and insert into the lipid membrane during ligand binding. The replacement of a hydrophobic residue at position 24 with a hydrophilic residue should interfere with this binding. STAC2 may thus fall into the category of proteins with atypical C1 domains that could be targeted with phorbol derivatives that do not stabilize membrane insertion. Our findings strengthen our insight into the structural basis and understanding of the interplay between the C1 domain, ligand and the membrane as a ternary complex. Citation Format: Adelle Abramovitz, Noemi Kedei, Peter M Blumberg. Understanding the structural requirements of C1 domains for phorbol ester binding, analysis of the atypical C1 domain of STAC2 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5344. doi:10.1158/1538-7445.AM2017-5344

The Ligand Binding Landscape of Diacylglycerol Kinases

Diacylglycerol kinases (DGKs) are integral components of signal transduction cascades that regulate cell biology through ATP-dependent phosphorylation of the lipid messenger diacylglycerol. Methods for direct evaluation of DGK activity in native biological systems are lacking and needed to study isoform-specific functions of these multidomain lipid kinases.Here, we utilize ATP acyl phosphate activity-based probes and quantitative mass spectrometry to define, for the first time, ATP and small-molecule binding motifs of representative members from all five DGK subtypes. We use chemical proteomics to discover an unusual binding mode for the DGKα inhibitor, ritanserin, including interactions at the atypical C1 domain distinct from the ATP binding region.Unexpectedly, deconstruction of ritanserin yielded afragment compound that blocks DGKα activitythrough a conserved binding mode and enhanced selectivity against the kinome. Collectively, our studies illustrate the power of chemical proteomics to profile protein-small molecule interactions of lipid kinases for fragment-based lead discovery.

Abstract 3928: Structural basis for the failure of the C1 domain of RasGRP2 to bind phorbol ester

Abstract C1 domains represent the recognition motif for the second messenger diacylglycerol (DAG) and for the phorbol esters in protein kinase C, RasGRP, and several other families of signaling proteins. In addition to such typical C1 domains, atypical C1 domains have been identified which possess varying levels of sequence and structural homology with the typical C1 domains but fail to bind DAG or phorbol ester. Among RasGRP family members, RasGRP1 and RasGRP3 possess typical C1 domains, whereas the C1 domain of RasGRP2 stands out as being atypical. In order to better understand the structural constraints for ligand binding, we have analyzed the basis for the failure of the C1 domain of RasGRP2 to bind ligands. Using site-directed mutagenesis, we have identified four critical amino acid residues responsible for the lack of phorbol ester sensitivity. We found that the C1 domain of RasGRP2 had weak binding affinity (Kd = 2890 ± 240 nM) in vitro for [3H]phorbol 12,13-dibutyrate ([3H]PDBu). Replacing all four of critical amino acid residues (Asn7, Ser8, Ala19 and Ile21) with the corresponding residues (Thr7, Tyr8, Gly19 and Leu21, respectively) of RasGRP1 resulted in potent [3H]PDBu binding affinity (Kd = 1.47 ± 0.03 nM) and translocation in response to PMA in the LNCaP cell line. The mutant C1 domains incorporating one to three of these critical residues showed intermediate binding and translocation properties. Of the four substitutions, S8Y made the greatest contribution. Phospholipid requirements for [3H]PDBu binding to the mutant C1 domains were determined; at constant total phospholipid, the requirement for the proportion of phosphatidylserine in phosphatidylserine : phosphatidylcholine mixtures decreased in going from the single S8Y mutant to the quadruple mutant. Binding activity for DAG was also restored in the mutant C1 domains, in parallel with that for phorbol ester. The full length RasGRP2 protein containing the mutated C1 domains likewise showed strong phorbol ester binding, albeit modestly weaker than that of the purified C1 domain (Kd = 8.2 ± 1.1 nM for the full length protein containing all four mutations) and displayed translocation in the LNCaP (human prostate adenocarcinoma) cells in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. A Rap1 GTPase pull-down assay was used to compare the guanine nucleotide exchange activity of wild type and mutated full length RasGRP2 in human embryonic kidney 293 (HEK 293) cells in the presence or absence of phorbol ester. These cells were transfected with either the wild type or the mutant RasGRP2. Consistent with the ability of phorbol ester to induce translocation of the full length RasGRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Citation Format: Agnes Czikora, Daniel J. Lundberg, Nancy E. Lewin, Noemi Kedei, Peter M. Blumberg. Structural basis for the failure of the C1 domain of RasGRP2 to bind phorbol ester. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3928. doi:10.1158/1538-7445.AM2015-3928

Diacylglycerol lactones targeting the structural features that distinguish the atypical C1 domains of protein kinase C ζ and ι from typical C1 domains.

To explore the feasibility of developing ligands targeted to the atypical C1 domains of protein kinase C ζ and ι, we have prepared diacylglycerol lactones substituted with hydrophilic groups on their side chains, which potentially could interact with the arginine residues that distinguish the atypical C1 domains of PKCζ and PKCι from typical C1 domains, and we have measured their binding to mutated versions of the C1b domain of PKCδ that incorporate one or more of these arginine residues. The most selective of the diacylglycerol lactones showed only a 10-fold reduction in binding affinity with the triple arginine mutant (N7R/S10R/L20R) compared to the wild-type, whereas phorbol 12,13-dibutyrate showed a 6000-fold loss of affinity. Molecular modeling confirms that these ligands are indeed able to interact with the arginine residues. Our results show that dramatic changes in selectivity can be obtained through appropriate substitution of diacylglycerol lactones.

Centralspindlin links the mitotic spindle to the plasma membrane during cytokinesis

At the end of cell division, cytokinesis splits the cytoplasm of nascent daughter cells and partitions segregated sister genomes. To coordinate cell division with chromosome segregation, the mitotic spindle controls cytokinetic events at the cell envelope. The spindle midzone stimulates the actomyosin-driven contraction of the cleavage furrow, which proceeds until the formation of a microtubule-rich intercellular bridge with the midbody at its centre. The midbody directs the final membrane abscission reaction and has been proposed to attach the cleavage furrow to the intercellular bridge. How the mitotic spindle is connected to the plasma membrane during cytokinesis is not understood. Here we identify a plasma membrane tethering activity in the centralspindlin protein complex, a conserved component of the spindle midzone and midbody. We demonstrate that the C1 domain of the centralspindlin subunit MgcRacGAP associates with the plasma membrane by interacting with polyanionic phosphoinositide lipids. Using X-ray crystallography we determine the structure of this atypical C1 domain. Mutations in the hydrophobic cap and in basic residues of the C1 domain of MgcRacGAP prevent association of the protein with the plasma membrane, and abrogate cytokinesis in human and chicken cells. Artificial membrane tethering of centralspindlin restores cell division in the absence of the C1 domain of MgcRacGAP. Although C1 domain function is dispensable for the formation of the midzone and midbody, it promotes contractility and is required for the attachment of the plasma membrane to the midbody, a long-postulated function of this organelle. Our analysis suggests that centralspindlin links the mitotic spindle to the plasma membrane to secure the final cut during cytokinesis in animal cells.

Abstract 3854: Identification of crucial residues at the rim of the binding cleft of the C1 domain of Vav1 that determine its ligand sensitivity

Abstract Vav1 is a guanine exchange factor (GEF) for the Rho family of GTPases. It plays a pivotal role in T-cell maturation and development, cytoskeleton organization, and oncogenic transformation. The GEF activity of Vav1 is regulated by several factors, including interaction between its catalytic DH domain and its C1 domain. Its C1 domain shows homology with “typical” C1 domains that are sensitive to the second messenger diacylglycerol (DAG) and phorbol esters (PEs), but it is classified as “atypical” based on its unresponsiveness to these ligands. However, crystallographic analysis has shown that, unlike atypical C1 domains (e.g. Raf1) which possess a distorted structure, the Vav1 C1 retains the geometry of the binding cavity. We hypothesized that residues in the vicinity of the binding pocket might interfere with ligand binding. Sequence alignment with typical C1 domains revealed six unique residues situated along the rim of the putative binding cleft in Vav1 C1: Glu9, Glu10, Pro11, Trp22, Thr24, Tyr26.To probe the role of these residues on DAG/PE sensitivity, we first mutated these sites in the potent PE-sensitive C1b domain of PKCΔ to that of the corresponding sites of Vav1 C1, and analyzed the potency of the mutants for PEs. In vitro binding assays showed that 5 of 6 single-site-mutations (except Trp22) caused significant but limited (10-15 fold) reduction in the binding affinity to phorbol 12,13-dibutyrate (PDBU). Introduction of multiple mutations further decreased the affinity, in a cumulative fashion, leading to no detectable binding in the quintuple mutant. Correspondingly, in vivo confocal microscopy revealed that double and triple GFP-tagged mutants showed much slower and weaker plasma membrane translocation in response to PE than did WT C1bΔ, whereas the quintuple mutation was completely unresponsive. Thus, the ligand-insensitivity of Vav1 C1 reflects the combined effects of Glu9, Glu10, Pro11, Thr24, Tyr26 rather than the effect of a single specific residue. Conversely, introducing “reverse” mutations (corresponding to the residues of PKCΔ C1b) into Vav1 C1 generate binding activity. The quintuple (PKCΔ-like) mutation restored the phorbol-ester-sensitivity of Vav1 C1 to the level of the potent PKCΔ C1b both in vitro and in vivo. In addition, the quintuple mutation conferred PE-sensitivity to the full length Vav1, as revealed by translocation studies. Computer modeling suggests that the presence of these residues confers on Vav1 C1 a hydrophilic surface at the tip of the binding cavity (as opposed to the rather lipophilic surface of PKCΔ C1b), thus impeding interactions with the membrane bilayer and hindering the formation of the ternary binding complex of ligand, receptor (C1) and membrane lipid. We speculate that targeting those unique hydrophilic residues with specific DAG/PE analogs may provide a rationale for selectively manipulating Vav1 function. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3854. doi:10.1158/1538-7445.AM2011-3854

Structure Basis and Unconventional Lipid Membrane Binding Properties of the PH-C1 Tandem of Rho Kinases

Rho kinase (ROCK), a downstream effector of Rho GTPase, is a serine/threonine protein kinase that regulates many crucial cellular processes via control of cytoskeletal structures. The C-terminal PH-C1 tandem of ROCKs has been implicated to play an autoinhibitory role by sequestering the N-terminal kinase domain and reducing its kinase activity. The binding of lipids to the pleckstrin homology (PH) domain not only regulates the localization of the protein but also releases the kinase domain from the close conformation and thereby activates its kinase activity. However, the molecular mechanism governing the ROCK PH-C1 tandem-mediated lipid membrane interaction is not known. In this study, we demonstrate that ROCK is a new member of the split PH domain family of proteins. The ROCK split PH domain folds into a canonical PH domain structure. The insertion of the atypical C1 domain in the middle does not alter the structure of the PH domain. We further show that the C1 domain of ROCK lacks the diacylglycerol/phorbol ester binding pocket seen in other canonical C1 domains. Instead, the inserted C1 domain and the PH domain function cooperatively in binding to membrane bilayers via the unconventional positively charged surfaces on each domain. Finally, the analysis of all split PH domains with known structures indicates that split PH domains represent a unique class of tandem protein modules, each possessing distinct structural and functional features.

Lck-Dependent Tyrosine Phosphorylation of Diacylglycerol Kinase α Regulates Its Membrane Association in T Cells

TCR engagement triggers phospholipase Cgamma1 activation through the Lck-ZAP70-linker of activated T cell adaptor protein pathway. This leads to generation of diacylglycerol (DAG) and mobilization of intracellular Ca(2+), both essential for TCR-dependent transcriptional responses. TCR ligation also elicits transient recruitment of DAG kinase alpha (DGKalpha) to the lymphocyte plasma membrane to phosphorylate DAG, facilitating termination of DAG-regulated signals. The precise mechanisms governing dynamic recruitment of DGKalpha to the membrane have not been fully elucidated, although Ca(2+) influx and tyrosine kinase activation were proposed to be required. We show that DGKalpha is tyrosine phosphorylated, and identify tyrosine 335 (Y335), at the hinge between the atypical C1 domains and the catalytic region, as essential for membrane localization. Generation of an Ab that recognizes phosphorylated Y335 demonstrates Lck-dependent phosphorylation of endogenous DGKalpha during TCR activation and shows that pY335DGKalpha is a minor pool located exclusively at the plasma membrane. Our results identify Y335 as a residue critical for DGKalpha function and suggest a mechanism by which Lck-dependent phosphorylation and Ca(2+) elevation regulate DGKalpha membrane localization. The concerted action of these two signals results in transient, receptor-regulated DGKalpha relocalization to the site at which it exerts its function as a negative modulator of DAG-dependent signals.

Role of the Diacylglycerol Kinase α-Conserved Domains in Membrane Targeting in Intact T Cells

Diacylglycerol kinase (DGK) phosphorylates diacylglycerol to phosphatidic acid, modifying the cellular levels of these two lipid mediators. Ten DGK isoforms, grouped into five subtypes, are found in higher organisms. All contain a conserved C-terminal domain and at least two cysteine-rich motifs of unknown function. DGKalpha is a type I enzyme that acts as a negative modulator of diacylglycerol-based signals during T cell activation. Here we studied the functional role of the DGKalpha domains using mutational analysis to investigate membrane binding in intact cells. We show that the two atypical C1 domains are essential for plasma membrane targeting of the protein in intact cells but unnecessary for catalytic activity. We also identify the C-terminal sequence of the protein as essential for membrane binding in a phosphatidic acid-dependent manner. Finally we demonstrate that, in the absence of the calcium binding domain, receptor-dependent translocation of the truncated protein is regulated by phosphorylation of Tyr(335). This functional study provides new insight into the role of the so-called conserved domains of this lipid kinase family and demonstrates the existence of additional domains that confer specific plasma membrane localization to this particular isoform.

CK2 Is a Component of the KSR1 Scaffold Complex that Contributes to Raf Kinase Activation

Kinase Suppressor of Ras (KSR) is a molecular scaffold that interacts with the core kinase components of the ERK cascade, Raf, MEK, and ERK and provides spatial and temporal regulation of Ras-dependent ERK cascade signaling. In this report, we identify the heterotetrameric protein kinase, casein kinase 2 (CK2), as a new KSR1-binding partner. Moreover, we find that the KSR1/CK2 interaction is required for KSR1 to maximally facilitate ERK cascade signaling and contributes to the regulation of Raf kinase activity. Binding of the CK2 holoenzyme is constitutive and requires the basic surface region of the KSR1 atypical C1 domain. Loss of CK2 binding does not alter the membrane translocation of KSR1 or its interaction with ERK cascade components; however, disruption of the KSR1/CK2 interaction or inhibition of CK2 activity significantly reduces the growth-factor-induced phosphorylation of C-Raf and B-Raf on the activating serine site in the negative-charge regulatory region (N-region). This decrease in Raf N-region phosphorylation further correlates with impaired Raf, MEK, and ERK activation. These findings identify CK2 as a novel component of the KSR1 scaffolding complex that facilitates ERK cascade signaling by functioning as a Raf family N-Region kinase.

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.