Critical Role of Trp-588 of Presynaptic Munc13-1 for Ligand Binding and Membrane Translocation.

Munc13-1 is a presynaptic active-zone protein essential for neurotransmitter release and presynaptic plasticity in the brain. This multidomain scaffold protein contains a C1 domain that binds to the activator diacylglycerol/phorbol ester. Although the C1 domain bears close structural homology with the C1 domains of protein kinase C (PKC), the tryptophan residue at position 22 (588 in the full-length Munc13-1) occludes the activator binding pocket, which is not the case for PKC. To elucidate the role of this tryptophan, we generated W22A, W22K, W22D, W22Y, and W22F substitutions in the full-length Munc13-1, expressed the GFP-tagged constructs in Neuro-2a cells, and measured their membrane translocation in response to phorbol ester treatment by imaging of the live cells using confocal microscopy. The extent of membrane translocation followed the order, wild-type > W22K > W22F > W22Y > W22A > W22D. The phorbol ester binding affinity of the wild-type Munc13-1C1 domain and its mutants was phosphatidylserine (PS)-dependent following the order, wild-type > W22K > W22A ≫ W22D in both 20% and 100% PS. Phorbol ester affinity was higher for Munc13-1 than the C1 domain. While Munc13-1 translocated to the plasma membrane, the C1 domain translocated to internal membranes in response to phorbol ester. Molecular dynamics (80 ns) studies reveal that Trp-22 is relatively less flexible than the homologous Trp-22 of PKCδ and PKCθ. Results are discussed in terms of the overall negative charge state of the Munc13-1C1 domain and its possible interaction with the PS-rich plasma membrane. This study shows that Trp-588 is an important structural element for ligand binding and membrane translocation in Munc13-1.

Biological activity of the bryostatin analog Merle 23 on mouse epidermal cells and mouse skin.

Bryostatin 1, a complex macrocyclic lactone, is the subject of multiple clinical trials for cancer chemotherapy. Although bryostatin 1 biochemically functions like the classic mouse skin tumor promoter phorbol 12-myristate 13-acetate (PMA) to bind to and activate protein kinase C, paradoxically, it fails to induce many of the typical phorbol ester responses, including tumor promotion. Intense synthetic efforts are currently underway to develop simplified bryostatin analogs that preserve the critical functional features of bryostatin 1, including its lack of tumor promoting activity. The degree to which bryostatin analogs maintain the unique pattern of biological behavior of bryostatin 1 depends on the specific cellular system and the specific response. Merle 23 is a significantly simplified bryostatin analog that retains bryostatin like activity only to a limited extent. Here, we show that in mouse epidermal cells the activity of Merle 23 was either similar to bryostatin 1 or intermediate between bryostatin 1 and PMA, depending on the specific parameter examined. We then examined the hyperplastic and tumor promoting activity of Merle 23 on mouse skin. Merle 23 showed substantially reduced hyperplasia and was not tumor promoting at a dose comparable to that for PMA. These results suggest that there may be substantial flexibility in the design of bryostatin analogs that retain its lack of tumor promoting activity. © 2016 Wiley Periodicals, Inc.

Charge density influences C1 domain ligand affinity and membrane interactions.

The C1 domain, which represents the recognition motif on protein kinase C for the lipophilic second messenger diacylglycerol and its ultrapotent analogues, the phorbol esters, has emerged as a promising therapeutic target for cancer and other indications. Potential target selectivity is markedly enhanced both because binding reflects ternary complex formation between the ligand, C1 domain, and phospholipid, and because binding drives membrane insertion of the C1 domain, permitting aspects of the C1 domain surface outside the binding site, per se, to influence binding energetics. Here, focusing on charged residues identified in atypical C1 domains which contribute to their loss of ligand binding activity, we showed that increasing charge along the rim of the binding cleft of the protein kinase C δ C1 b domain raises the requirement for anionic phospholipids. Correspondingly, it shifts the selectivity of C1 domain translocation to the plasma membrane, which is more negatively charged than internal membranes. This change in localization is most pronounced in the case of more hydrophilic ligands, which provide weaker membrane stabilization than do the more hydrophobic ligands and thus contributes an element to the structure-activity relations for C1 domain ligands. Coexpressing pairs of C1-containing constructs with differing charges each expressing a distinct fluorescent tag provided a powerful tool to demonstrate the effect of increasing charge in the C1 domain.

Synthesis, biological, and biophysical studies of DAG-indololactones designed as selective activators of RasGRP.

The development of selective agents capable of discriminating between protein kinase C (PKC) isoforms and other diacylglycerol (DAG)-responsive C1 domain-containing proteins represents an important challenge. Recent studies have highlighted the role that Ras guanine nucleotide-releasing protein (RasGRP) isoforms play both in immune responses as well as in the development of prostate cancer and melanoma, suggesting that the discovery of selective ligands could have potential therapeutic value. Thus far, the N-methyl-substituted indololactone 1 is the agonist with the highest reported potency and selectivity for RasGRP relative to PKC. Here we present the synthesis, binding studies, cellular assays and biophysical analysis of interactions with model membranes of a family of regioisomers of 1 (compounds 2-5) that differ in the position of the linkage between the indole ring and the lactone moiety. These structural variations were studied to explore the interaction of the active complex (C1 domain-ligand) with cellular membranes, which is believed to be an important factor for selectivity in the activation of DAG-responsive C1 domain containing signaling proteins. All compounds were potent and selective activators of RasGRP when compared to PKCα with selectivities ranging from 6 to 65 fold. However, the parent compound 1 was appreciably more selective than any of the other isomers. In intact cells, modest differences in the patterns of translocation of the C1 domain targets were observed. Biophysical studies using giant vesicles as model membranes did show substantial differences in terms of molecular interactions impacting lipid organization, dynamics and membrane insertion. However, these differences did not yield correspondingly large changes in patterns of biological response, at least for the parameters examined.

Evidence of an evolutionarily conserved LMBR1 domain-containing protein that associates with endocytic cups and plays a role in cell migration in dictyostelium discoideum.

The ampA gene plays a role in Dictyostelium discoideum cell migration. Loss of ampA function results in reduced ability of growing cells to migrate to folic acid and causes small plaques on bacterial lawns, while overexpression of AmpA results in a rapid-migration phenotype and correspondingly larger plaques than seen with wild-type cells. To help understand how the ampA gene functions, second-site suppressors were created by restriction enzyme-mediated integration (REMI) mutagenesis. These mutants were selected for their ability to reduce the large plaque size of the AmpA overexpresser strain. The lmbd2B gene was identified as a suppressor of an AmpA-overexpressing strain. The lmbd2B gene product belongs to the evolutionarily conserved LMBR1 protein family, some of whose known members are endocytic receptors associated with human diseases, such as anemia. In order to understand lmbd2B function, mRFP fusion proteins were created and lmbd2B knockout cell lines were established. Our findings indicate that the LMBD2B protein is found associated with endocytic cups. It colocalizes with proteins that play key roles in endocytic events and is localized to ruffles on the dorsal surfaces of growing cells. Vegetative lmbd2B-null cells display defects in cell migration. These cells have difficulty sensing the chemoattractant folic acid, as indicated by a decrease in their chemotactic index. lmbd2B-null cells also appear to have difficulty establishing a front/back orientation to facilitate migration. A role for lmbd2B in development is also suggested. Our research gives insight into the function of a previously uncharacterized branch of the LMBR1 family of proteins. We provide evidence of an LMBR1 family plasma membrane protein that associates with endocytic cups and plays a role in chemotaxis.

The adhesion modulation protein, AmpA localizes to an endocytic compartment and influences substrate adhesion, actin polymerization and endocytosis in vegetative Dictyostelium cells.

BACKGROUND: AmpA is a secreted 24Kd protein that has pleiotropic effects on Dictyostelium development. Null mutants delay development at the mound stage with cells adhering too tightly to the substrate. Prestalk cells initially specify as prespore cells and are delayed in their migration to the mound apex. Extracellular AmpA can rescue these defects, but AmpA is also necessary in a cell autonomous manner for anterior like cells (ALCs) to migrate to the upper cup. The ALCs are only 10% of the developing cell population making it difficult to study the cell autonomous effect of AmpA on the migration of these cells. AmpA is also expressed in growing cells, but, while it contains a hydrophobic leader sequence that is cleaved, it is not secreted from growing cells. This makes growing cells an attractive system for studying the cell autonomous function of AmpA. RESULTS: In growing cells AmpA plays an environment dependent role in cell migration. Excess AmpA facilitates migration on soft, adhesive surfaces but hinders migration on less adhesive surfaces. AmpA also effects the level of actin polymerization. Knockout cells polymerize less actin while over expressing cells polymerize more actin than wild type. Overexpression of AmpA also causes an increase in endocytosis that is traced to repeated formation of multiple endocytic cups at the same site on the membrane. Immunofluorescence analysis shows that AmpA is found in the Golgi and colocalizes with calnexin and the slow endosomal recycling compartment marker, p25, in a perinuclear compartment. AmpA is found on the cell periphery and is endocytically recycled to the perinuclear compartment. CONCLUSION: AmpA is processed through the secretory pathway and traffics to the cell periphery where it is endocytosed and localizes to what has been defined as a slow endosomal recycling compartment. AmpA plays a role in actin polymerization and cell substrate adhesion. Additionally AmpA influences cell migration in an environment dependent manner. Wild type cells show very little variation in migration rates under the different conditions examined here, but either loss or over expression of AmpA cause significant substrate and environment dependent changes in migration.

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.

A SAP domain-containing protein shuttles between the nucleus and cell membranes and plays a role in adhesion and migration in D. discoideum.

The AmpA protein reduces cell adhesion, thereby influencing cell migration in Dictyostelium. To understand how ampA influences cell migration, second site suppressors of an AmpA overexpressing cell line were created by REMI mutagenesis. Mutant candidates were identified by their ability to suppress the large plaques that the AmpA overexpressing cells form on bacterial lawns as a result of their increased rate of migration. One suppressor gene, sma, encodes an uncharacterized protein, which contains a SAP DNA-binding domain and a PTEN-like domain. Using sma gene knockouts and Sma-mRFP expressing cell lines, a role for sma in influencing cell migration was uncovered. Knockouts of the sma gene in a wild-type background enhanced chemotaxis. An additional role for Sma in influencing cell-cell adhesion was also demonstrated. Sma protein transitions between cytosolic and nuclear localizations as a function of cell density. In growing cells migrating to folic acid it is localized to regions of actin polymerization and absent from the nucleus. A role for Sma in influencing ampA mRNA levels is also demonstrated. Sma additionally appears to be involved in ampA pathways regulating cell size, actin polymerization, and cell substrate adhesion. We present insights to the SAP domain-containing group of proteins in Dictyostelium and provide evidence of a role for a SAP domain-containing protein shuttling from the nucleus to sites of actin polymerization during chemotaxis to folic acid and influencing the efficiency of migration.

RasGRPs are targets of the anti-cancer agent ingenol-3-angelate.

Ingenol-3-angelate (I3A) is a non-tumor promoting phorbol ester-like compound identified in the sap of Euphoria peplus. Similar to tumor promoting phorbol esters, I3A is a diacylglycerol (DAG) analogue that binds with high affinity to the C1 domains of PKCs, recruits PKCs to cellular membranes and promotes enzyme activation. Numerous anti-cancer activities have been attributed to I3A and ascribed to I3A's effects on PKCs. We show here that I3A also binds to and activates members of the RasGRP family of Ras activators leading to robust elevation of Ras-GTP and engagement of the Raf-Mek-Erk kinase cascade. In response to I3A, recombinant proteins consisting of GFP fused separately to full-length RasGRP1 and RasGRP3 were rapidly recruited to cell membranes, consistent with direct binding of the compound to RasGRP's C1 domain. In the case of RasGRP3, IA3 treatment led to positive regulatory phosphorylation on T133 and activation of the candidate regulatory kinase PKCδ. I3A treatment of select B non-Hodgkin's lymphoma cell lines resulted in quantitative and qualitative changes in Bcl-2 family member proteins and induction of apoptosis, as previously demonstrated with the DAG analogue bryostatin 1 and its synthetic analogue pico. Our results offer further insights into the anticancer properties of I3A, support the idea that RasGRPs represent potential cancer therapeutic targets along with PKC, and expand the known range of ligands for RasGRP regulation.

Ndm, a coiled-coil domain protein that suppresses macropinocytosis and has effects on cell migration.

The ampA gene has a role in cell migration in Dictyostelium discoideum. Cells overexpressing AmpA show an increase in cell migration, forming large plaques on bacterial lawns. A second-site suppressor of this ampA-overexpressing phenotype identified a previously uncharacterized gene, ndm, which is described here. The Ndm protein is predicted to contain a coiled-coil BAR-like domain-a domain involved in endocytosis and membrane bending. ndm-knockout and Ndm-monomeric red fluorescent protein-expressing cell lines were used to establish a role for ndm in suppressing endocytosis. An increase in the rate of endocytosis and in the number of endosomes was detected in ndm(-) cells. During migration ndm(-) cells formed numerous endocytic cups instead of the broad lamellipodia structure characteristic of moving cells. A second lamellipodia-based function-cell spreading-was also defective in the ndm(-) cells. The increase in endocytosis and the defect in lamellipodia formation were associated with reduced chemotaxis in ndm(-) cells. Immunofluorescence results and glutathione S-transferase pull-down assays revealed an association of Ndm with coronin and F-actin. The results establish ndm as a gene important in regulating the balance between formation of endocytic cups and lamellipodia structures.