β2-Chimaerin Is a High Affinity Receptor for the Phorbol Ester Tumor Promoters

Abstract

␤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...

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