β2-Chimaerin is a novel target for diacylglycerol: Binding properties and changes in subcellular localization mediated by ligand binding to its C1 domain

Abstract

The members of the chimaerin family of Rac-GTPase-activating proteins possess a single C1 domain with high homology to those present in protein kinase C (PKC) isozymes. This domain in PKCs is involved in phorbol ester and diacylglycerol (DAG) binding. We previously have demonstrated that one of the chimaerin isoforms, ␤2-chimaerin, binds phorbol esters with high affinity. In this study we analyzed the properties of ␤2-chimaerin as a DAG receptor by using a series of conformationally constrained cyclic DAG analogues (DAG lactones) as probes. We identified analogs that bind to ␤2-chimaerin with more than 100-fold higher affinity than 1-oleoyl-2-acetylglycerol. The potencies of these analogs approach those of the potent phorbol ester tumor promoters. The different DAG lactones show some selectivity for this novel receptor compared with PKC␣. Cellular studies revealed that these DAG analogs induce translocation of ␤2-chimaerin from cytosolic (soluble) to particulate fractions. Using green fluorescent protein-fusion proteins for ␤2-chimaerin we determined that this novel receptor translocates to the perinuclear region after treatment with DAG lactones. Binding and translocation were prevented by mutation of the conserved Cys-246 in the C1 domain. The structural homology between the C1 domain of ␤2-chimaerin and the C1b domain of PKC␦ also was confirmed by modeling analysis. Our results demonstrate that ␤2-chimaerin is a high affinity receptor for DAG through binding to its C1 domain and supports the emerging concept that multiple pathways transduce signaling through DAG and the phorbol esters. Signaling in response to the second messenger diacylgycerol (DAG) is thought to proceed through the activation of protein kinase C (PKC) isozymes (1, 2). Binding of this lipid second messenger and its related analogs, the phorbol esters, occurs at the C1 domains (also called cysteine-rich regions or zinc fingers) present in the classical PKCs (PKC␣, ␤I, ␤II, and ␥) and novel PKCs (PKC␦, , , and ). This 50-to 51-aa domain, which is present in tandem in these PKC isozymes, possesses the motif HX 12 CX 2 CX n CX 2 CX 4 HX 2 CX 7 C, where H is histidine, C is cysteine, X is any other amino acid, and n is 13-14 (3-6). The phorbol ester receptor family has expanded with the discovery of the chimaerins. Unlike PKCs, the chimaerins do not possess a functional kinase domain but they are GTPase-activating proteins for Rac, a small GTP binding protein of the Ras superfamily (7). Four chimaerin isoforms (␣1-or n-, ␣2-, ␤1-, and ␤2-chimaerin) have been identified to date, all of them possessing a single C1 domain with approximately 40% homology to those present in PKCs (7-10). It is therefore predictable that the biological responses of the phorbol esters and those mediated by DAG signaling could involve the activation of PKC-independent pathways.We previously have reported that ␣1-and ␤2-chimaerin are indeed high affinity receptors for the phorbol esters and also for the bryostatins, macrocyclic lactones with antitumor properties (11,12). ...

The diacylglycerol-binding protein α1-chimaerin regulates dendritic morphology

“…To this end we generated three mutant proteins: one containing a point mutation in the C1 domain that disrupts phorbol-ester binding (C114A) (23); one containing a point mutation in the GAP domain (R179G) that abolishes Rac-GAP activity (21,24); and a deletion mutant in which the entire GAP domain was deleted (⌬GAP). To confirm Rac-GAP activity of the two point mutants we performed GTPase pull-down assays with the Rac effector (CRIB) domain of the serine͞threonine kinase PAK1, which selectively affinity purifies the GTP-bound form of Rac (Fig.…”

Directional sensing of a phorbol ester gradient requires CD44 and is regulated by CD44 phosphorylation

“…The identities of the Ser316 kinase and Ser325 phosphatase acting downstream of PKC have yet to be identified but these studies have ruled out PKA and mitogen-activated protein kinases in this process. In addition to the phorbol ester-binding PKC isoenzymes, PMA has been reported to bind other receptors such as the chimaerins (Caloca et al, 1999), Ras guanyl-releasing protein (ElShemerly et al, 1997;Ebinu et al, 1998) and protein kinase Ds (Van Lint et al, 2002). Importantly, this study and previous studies (Legg et al, 2002) have demonstrated that the effects of PMA treatment on CD44 Ser316 phosphorylation and Ser325 dephosphorylation are mediated via activation of PKC as the PKC inhibitors Ro-31 and Bis-I are effective in blocking these phosphorylation/dephosphorylation events.…”

Gonadotropin-releasing Hormone-induced Activation of Diacylglycerol Kinase-ζ and Its Association with Active c-Src

“…DAG can act as a membrane recruitment signal and activator for a series of signaling proteins, e.g. it has been shown that plasma membrane DAG is vital for the activation of some PKC isoforms (38), UNC-13 (39), RasGRP (40), and chimeras (41). The DAG-mediated control of these signaling proteins underlies its role in affecting cell proliferation, differentiation, and apoptosis.…”