2-chimaerin, a member of the GTPase-activating proteins for the small GTP-binding protein p21Rac, possesses a single cysteine-rich domain with high homology to those implicated in phorbol ester and diacylglycerol binding in protein kinase C (PKC) isozymes. We have expressed 2-chimaerin in Sf9 insect cells using the baculovirus expression system and determined that, like PKCs, 2-chimaerin binds phorbol esters with high affinity in the presence of phosphatidylserine as a cofactor. Scatchard plot analysis using the radioligand [ 3 H]phorbol 12,13-dibutyrate revealed a dissociation constant of 1.9 ؎ 0.2 nM for 2-chimaerin. Likewise, 2-chimaerin is a high affinity receptor for the bryostatins, a class of atypical PKC activators. A detailed comparison of structure-activity relations using several phorbol ester analogs revealed striking differences in binding recognition between 2-chimaerin and PKC␣. Although the diacylglycerol 1-oleoyl-2-acetylglycerol binds with similar potency to both 2-chimaerin and PKC␣, the mezerein analog thymeleatoxin has 56-fold less affinity for binding to 2-chimaerin. To establish whether 2-chimaerin responds to phorbol esters in cellular systems, we overexpressed 2-chimaerin in COS-7 cells and monitored its subcellular distribution after phorbol ester treatment. Interestingly, as described previously for PKC isozymes, 2-chimaerin translocates from cytosolic to particulate fractions as a consequence of phorbol ester treatment. Our results demonstrate that 2-chimaerin is a novel target for the phorbol ester tumor promoters. The expansion of the family of phorbol ester receptors strongly suggests a potential for the "nonkinase" receptors as cellular mediators of the phorbol ester responses.The phorbol esters and related diterpenes are natural compounds that are used widely as tumor promoters in animal models. The search for receptors for the phorbol esters led to the identification of protein kinase C (PKC) 1 as their target (1-5). The complexity and heterogeneity in the biology of phorbol esters suggested the existence of multiple receptors, and 11 PKC isoforms have been identified so far: classic or calciumdependent isozymes (PKC ␣, 1, 2, and ␥), novel or calciumindependent isozymes (PKC ␦, ⑀, , , and ), and atypical isozymes (PKC and ) (6, 7). The last group is unresponsive to the phorbol esters. DAG, the postulated endogenous activator of PKC, competes with phorbol esters for binding to PKC in in vitro assays, although its binding potency is lower than that of the most commonly used phorbol esters (8). The cysteine-rich domains present at the NH 2 -terminal region of the PKCs are the binding sites for DAG/phorbol esters (9 -13). Each of these 50-or 51-amino acid domains possesses the motif HX 12 CX 2 CX 13/14 CX 2 CX 4 HX 2 CX 7 C, where H is histidine, C is cysteine, and X is any other amino acid. This motif is duplicated in tandem in both the classic and novel PKCs and is present only once in the atypical PKCs. The cysteine-rich domains have been implicated in the associati...
Purpose-This article makes a case for the importance of brokering future learning opportunities to youth as a programmatic goal for informal learning organizations. Such brokering entails engaging in practices that connect youth to events, programs, internships, individuals and institutions related to their interests to support them beyond the window of a specific program or event. Brokering is especially critical for youth who are new to an area of interest: it helps them develop both a baseline understanding of the information landscape and a social network that will respond to their needs as they pursue various goals. The paper aims to describe three critical levers for brokering well in informal settings: creating learning environments that allow trust to form between youth and educators and enable educators to develop an understanding of a young person's interests, needs and goals; attending to a young person's tendency (or not) to reach out to educators after a program is over to solicit assistance; and enabling potential brokers to efficiently locate appropriate future learning opportunities for each young person who approaches them. The authors also include a set of program practices for providers who wish to increase their brokering impact, as well as recommendations geared primarily toward organization leaders. The authors hope that this paper brings clarity and enhanced significance to the practice of brokering as a strategy to support youth pathways toward meaningful futures. Design/methodology/approach-Insights presented here are the result of a participatory knowledge building and sharing process with a community of after-school providers known as the Mozilla Hive NYC Learning Network. The topic of discussion was how these providers might continue to support young people in their intensive project-based programs after the program was over. The authors of this article, acting as embedded research partners to Hive NYC, contributed insights to these discussions based on ethnographic fieldwork and case studies of high-school-age youth in the Hive NYC context. Findings-The authors articulate a set of brokering practices and a conceptual model that communicates how brokering might lead to valued long-term outcomes for youth, including increased social capital. Originality/value-The intent is that information and perspectives from this article will inform youth-serving practice and serve as a catalyst for further conversations and activities geared toward promoting youth pathways of learning and identity development.
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