Background: The C1 domain of Vav1 retains a three-dimensional structure consistent with phorbol ester binding but nevertheless does not bind. Results: Five residues render the C1 domain less lipophilic and interfere with its binding. Conclusion: The C1 domain of Vav1 illustrates a novel mechanism rendering the C1 domain "atypical." Significance: Ligands exploiting the specific amino acid differences may selectively target Vav1.

The bryostatins are a group of twenty macrolides isolated by Pettit and coworkers from the marine organism Bugula neritina. Bryostatin 1, the flagship member of the family, has been the subject of intense chemical and biological investigations due to its remarkably diverse biological activities, including promising indications as therapy for cancer, Alzheimer’s disease, and HIV. Other bryostatins, however, have attracted far less attention, most probably due to their relatively low natural abundance and associated scarcity of supply. Among all macrolides in this family, bryostatin 7 is biologically the most potent PKC (protein kinase C) ligand (in terms of binding affinity) and also the first bryostatin to be synthesized in the laboratory. Nonetheless, almost no biological studies have been carried out on this agent. We describe herein the total synthesis of bryostatin 7 based on our pyran annulation technology, which allows for the first detailed biological characterizations of bryostatin 7 with side-by-side comparisons to bryostatin 1. The results suggest that the more easily synthesized and less lipophilic bryostatin 7 may be an effective surrogate for bryostatin 1.

The C1 domain, which represents the recognition motif on protein kinase C for the lipophilic second messenger diacylglycerol and its ultrapotent analog 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 ligand, the 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 show that increasing charge along the rim of the binding cleft of the protein kinase C δ C1b 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. Co-expressing 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.

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