Although multiple natural products are potent ligands for the diacylglycerol binding C1 domain of protein kinase C (PKC), RasGRP, and related targets, the high conservation of C1 domains has impeded the development of selective ligands. We characterized here a diacylglycerol-lactone, 130C037, emerging from a combinatorial chemical synthetic strategy, which showed substantial selectivity. 130C037 gave shallow binding curves for PKC isoforms ␣, ␤, ␥, ␦, and ⑀, with apparent K i values ranging from 340 nM for PKC␣ to 29 nM for PKC⑀. When binding to isolated C1 domains of PKC␣ and -␦, 130C037 showed good affinity (K i ‫؍‬ 1.78 nM) only for ␦C1b, whereas phorbol 12,13-dibutyrate showed affinities within 10-fold for all. In LNCaP cells, 130C037 likewise selectively induced membrane translocation of ␦C1b. 130C037 bound intact RasGRP1 and RasGRP3 with K i values of 3.5 and 3.8 nM, respectively, reflecting 8-and 90-fold selectivity relative to PKC⑀ and PKC␣. By Western blot of Chinese hamster ovary cells, 130C037 selectively induced loss from the cytosol of RasGRP3 (ED 50 ‫؍‬ 286 nM), partial reduction of PKC⑀ (ED 50 > 10 M), and no effect on PKC␣. As determined by confocal microscopy in LNCaP cells, 130C037 caused rapid translocation of RasGRP3, limited slow translocation of PKC⑀, and no translocation of PKC␣. Finally, 130C037 induced Erk phosphorylation in HEK-293 cells ectopically expressing RasGRP3 but not in control cells, whereas phorbol ester induced phosphorylation in both. The properties of 130C037 provide strong proof of principle for the feasibility of developing ligands with selectivity among C1 domain-containing therapeutic targets. Diacylglycerol (DAG)1 is a lipid second messenger, produced through hydrolysis of phosphatidylinositol 4,5-bisphosphate following the activation of receptor-coupled phospholipase C or indirectly from phosphatidylcholine via phospholipase D (1). Most but not all effects of DAG reflect its interaction with proteins containing C1 domains, resulting in their activation and/or driving their membrane translocation. Reflecting the importance and diversity of its downstream effectors, DAG is involved in signal transduction of numerous physiological and pathological processes, including proliferation, differentiation, apoptosis, angiogenesis, and drug resistance (2). These functions have focused attention on C1 domain-containing proteins as molecular targets for cancer chemotherapy (3).The interaction between DAG and its receptors is typically mediated by a DAG-responsive motif called a "C1 domain" (4). The highly conserved C1 domain (ϳ50 amino acids) is a cysteine-rich zinc finger structure (5) that was first identified in protein kinase C (PKC) as the interaction site for DAG and the phorbol esters (6). The PKC family of serine/threonine protein kinases comprises the best studied mediators of DAG signaling. 8 of its 11 family members have DAG-responsive C1 domains: (i) the conventional PKCs (␣, ␤I, ␤II, and ␥) and (ii) the novel PKCs (␦, ⑀, , and ). Both the classic and novel PKCs conta...

We describe the development of a next-generation mentoring survey drawing from prior surveys, capital theory, and critical race theory, with the goal of improving mentoring for students from underrepresented groups in science, technology, engineering, and mathematics. This survey focused on deaf mentees. The results show that the mentor’s cultural competence affected mentoring experiences.

Scientists are shaped by their unique life experiences and bring these perspectives to their research. Diversity in life and cultural experiences among scientists, therefore, broadens research directions and, ultimately, scientific discoveries. Deaf individuals, for example, have successfully contributed their unique perspectives to scientific inquiry. However, deaf individuals still face challenges in university science education. Most deaf students in science, technology, engineering, and mathematics (STEM) disciplines interact with faculty who have little to no experience working with deaf individuals and who often have preconceptions or simply a lack of knowledge about deaf individuals. In addition to a lack of communication access, deaf students may also feel unwelcome in STEM, as do other underrepresented groups. In this essay, we review evidence from the literature and, where data are lacking, contribute the expert opinions of the authors, most of whom are deaf scientists themselves, to identify strategies to best support deaf students in university STEM education. We describe the journey of a hypothetical deaf student and methods for faculty to create a welcoming environment. We describe and provide recommendations for classroom seating and layout, accommodations, teaching strategies, and research mentoring. We also discuss the importance of including deaf scientists in research about deaf individuals.

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The role of the protein kinase C (PKC) family of serine/ threonine kinases in cellular differentiation, proliferation, apoptosis, and other responses makes them attractive therapeutic targets. The activation of PKCs by ligands in vivo varies depending upon cell type; therefore, methods are needed to screen the potency of PKCs in this context. Here we describe a genetically encoded chimera of native PKC␦ fused to yellow-and cyanshifted green fluorescent protein, which can be expressed in mammalian cells. This chimeric protein kinase, CY-PKC␦, retains native or near-native activity in the several biological and biochemical parameters that we tested. Binding assays showed that CY-PKC␦ and native human PKC␦ have similar binding affinity for phorbol 12,13-dibutyrate. Analysis of translocation by Western blotting and confocal microscopy showed that CY-PKC␦ translocates from the cytosol to the membrane upon treatment with ligand, that the translocation has similar dose dependence as that of endogenous PKC␦, and that the pattern of translocation is indistinguishable from that of the green fluorescent protein-PKC␦ fusion well characterized from earlier studies. Treatment with phorbol ester of cells expressing CY-PKC␦ resulted in a dose-dependent increase in FRET that could be visualized in situ by confocal microscopy or measured fluorometrically. By using this construct, we were able to measure the kinetics and potencies of 12 known PKC ligands, with respect to CY-PKC␦, in the intact cell. The CY-PKC␦ chimera and the in vivo assays described here therefore show potential for high throughput screening of prospective PKC␦ ligands within the context of cell type.The members of the protein kinase C (PKC) 1 family of serine/ threonine kinases represent critical signaling molecules in the cell. PKCs are activated by the second messenger sn-1,2-diacylglycerol (DAG) or by their ultrapotent analogues, the phorbol esters. Because PKCs activate downstream cellular pathways that regulate cell proliferation, apoptosis, differentiation, and other responses, they are important therapeutic targets. Indeed, a number of compounds targeting protein kinase C are currently at different stages of drug development. Examples include LY333531, being evaluated for diabetic retinopathy and anti-angiogenesis (1, 2), prostratin and dPP, being evaluated for AIDS chemotherapy (3, 4), and bryostatin 1 and ingenol 3-angelate, being evaluated as cancer chemotherapeutic agents (5-7).The known isoforms of protein kinase C include the classical PKCs (␣, ␤ I , ␤ II , and ␥), which are calcium-dependent; the novel PKCs (␦, ⑀, , and ), which are calcium-independent, and the atypical PKCs ( and ), which are not activated by DAG or the phorbol esters (8, 9). The classical and novel PKCs each contain one or more highly conserved C1 domains, zinc finger motifs that act as the binding site for DAG and the phorbol esters (10, 11).Biochemical assays of PKC activation have limited predictive value for the understanding of structure-activity relationships because of ex...

The C1 domain represents the recognition module for diacylglycerol and phorbol esters in protein kinase C, Ras guanine nucleotide releasing protein (RasGRP), and related proteins. 3 H]phorbol 12,13-dibutyrate binding was determined; it decreased in going from the single S8Y mutant to the quadruple mutant. The full-length RasGRP2 protein with the mutated C1 domains also showed strong phorbol ester binding, albeit modestly weaker than that of the C1 domain alone (K d ‫؍‬ 8.2 ؎ 1.1 nM for the full-length protein containing all four mutations), and displayed translocation in response to phorbol ester. RasGRP2 is a guanyl exchange factor for Rap1. Consistent with the ability of phorbol ester to induce translocation of the full-length Ras-GRP2 with the mutated C1 domain, phorbol ester enhanced the ability of the mutated RasGRP2 to activate Rap1. Modeling confirmed that the four mutations helped the binding cleft maintain a stable conformation. RasGRP2 is exceptional in that its

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.

Evidence that the ligand binding site of TRPV1 lies on the inner face of the plasma membrane and that much of the TRPV1 itself is localized to internal membranes suggests that the rate of ligand entry into the cell may be an important determinant of the kinetics of ligand action. In this study, we synthesized a BODIPY TR-labeled fluorescent capsaicin analog (CHK-884) so that we could directly measure ligand entry. We report that CHK-884 penetrated only slowly into Chinese hamster ovary (CHO) cells expressing rat TRPV1, with a t 1/2 of 30 Ϯ 4 min, and localized in the endoplasmic reticulum and Golgi. Although CHK-884 was only weakly potent for TRPV1 binding (K i ϭ 6400 Ϯ 230 nM), it was appreciably more potent when assayed by intracellular calcium imaging and was 3.2-fold more potent with a 1-h incubation time (37 nM) than with a 5-min incubation time. Olvanil, a highly lipophilic vanilloid, yielded an EC 50 of 4.3 nM upon intracellular calcium imaging with an incubation time of 1 h, compared with an EC 50 value of 29.5 nM for calcium imaging assayed at 5 min. Likewise, the antagonist 5-iodoresiniferatoxin (5-iodo-RTX) displayed a K i of 4.2 pM if incubated with CHO-TRPV1 cells for 2 h before addition of capsaicin compared with 1.5 nM if added simultaneously. We conclude that some vanilloids may have slow kinetics of uptake; this slow uptake may affect assessment of structure activity relations and may represent a significant factor for vanilloid drug design.TRPV1 is a central nociceptor mediating response to vanilloids (such as capsaicin and RTX), heat, low pH, and endogenous ligands (Szallasi and Blumberg, 1989;Caterina et al., 1997;Zygmunt et al., 1999;Hwang et al., 2000;Gavva et al., 2004). In addition, it has a prominent role in the functioning of C-fiber sensory neurons, thus becoming a promising therapeutic target for chronic pain, bladder hyperreflexia, pruritus, diabetic neuropathy, postherpetic neuropathy, or cough (Robbins, 2000;Morice and Geppetti, 2004).In this study, we were concerned with the influence of the rate of vanilloid penetration into the cell on apparent vanilloid activity. It is clear that TRPV1 shows complicated cellular localization. Contrary to the expectation that TRPV1 should be localized at the plasma membrane, most seems to be located at internal membranes (Olah et al., 2001). Consistent with this pattern of localization, multiple research groups have shown that TRPV1 can function to release calcium from endoplasmic stores as well as permit calcium entry from outside the cell (Eun et al., 2001;Marshall et al., 2003). Vanilloids obviously need to penetrate into the cell to gain access to the endoplasmic reticulum localized TRPV1.For the TRPV1 located at the plasma membrane, the original view was, likewise, that the vanilloid binding site of -2004-000-10132-0