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C1 domains, the recognition motif of the second messenger diacylglycerol and of the phorbol esters, are classified as typical (ligand-responsive) or atypical (not ligand-responsive). The C1 domain of Vav1, a guanine nucleotide exchange factor, plays a critical role in regulation of Vav activity through stabilization of the Dbl homology domain, which is responsible for exchange activity of Vav. Although the C1 domain of Vav1 is classified as atypical, it retains a binding pocket geometry homologous to that of the typical C1 domains of PKCs. This study clarifies the basis for its failure to bind ligands. Substituting Vav1-specific residues into the C1b domain of PKCδ, we identified five crucial residues (Glu(9), Glu(10), Thr(11), Thr(24), and Tyr(26)) along the rim of the binding cleft that weaken binding potency in a cumulative fashion. Reciprocally, replacing these incompatible residues in the Vav1 C1 domain with the corresponding residues from PKCδ C1b (δC1b) conferred high potency for phorbol ester binding. Computer modeling predicts that these unique residues in Vav1 increase the hydrophilicity of the rim of the binding pocket, impairing membrane association and thereby preventing formation of the ternary C1-ligand-membrane binding complex. The initial design of diacylglycerol-lactones to exploit these Vav1 unique residues showed enhanced selectivity for C1 domains incorporating these residues, suggesting a strategy for the development of ligands targeting Vav1.

Phorbol 12-myristate 13-acetate (PMA) and bryostatin 1 are both potent protein kinase C (PKC) activators. In LNCaP human prostate cancer cells, PMA induces tumor necrosis factor alpha (TNFα) secretion and inhibits proliferation; bryostatin 1 does not, and indeed blocks the response to PMA. This difference has been attributed to bryostatin 1 not localizing PKCδ to the plasma membrane. Since phorbol ester lipophilicity influences PKCδ localization, we have examined in LNCaP cells a series of phorbol esters and related derivatives spanning some eight logs in lipophilicity (logP) to see if any behave like bryostatin 1. The compounds showed marked differences in their effects on proliferation and TNFα secretion. For example, maximal responses for TNFα secretion relative to PMA ranged from 97 % for octyl-indolactam V to 24 % for phorbol 12,13-dibenzoate. Dose-response curves ranged from monophasic for indolactam V to markedly biphasic for sapintoxin D. The divergent patterns of response, however, correlated neither to lipophilicity, to plasma membrane translocation of PKCδ, nor to the ability to interact with model membranes. In U937 human leukemia cells, a second system in which PMA and bryostatin 1 have divergent effects, viz. PMA but not bryostatin 1 inhibits proliferation and induces attachment, all the compounds acted like PMA for proliferation, but several induced a reduced level or a biphasic dose-response curve for attachment. We conclude that active phorbol esters are not all equivalent. Depending on the system, some might partially resemble bryostatin 1 in their behavior; this encourages the concept that bryostatin-like behavior may be obtained from other structural templates., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Abstract: Bryostatin 1 has attracted considerable attention both as a cancer chemotherapeutic agent and for its unique activity. Although it functions, like phorbol esters, as a potent protein kinase C (PKC) activator, it paradoxically antagonizes many phorbol ester responses in cells. Because of its complex structure, little is known of its structure-function relations. Merle 23 is a synthetic derivative, differing from bryostatin 1 at only four positions. However, in U-937 human leukemia cells, Merle 23 behaves like a phorbol ester and not like bryostatin 1. Here, we characterize the behavior of Merle 23 in the human prostate cancer cell line LNCaP. In this system, bryostatin 1 and phorbol ester have contrasting activities, with the phorbol ester but not bryostatin 1 blocking cell proliferation or tumor necrosis factor alpha secretion, among other responses. We show that Merle 23 displays a highly complex pattern of activity in this system. Depending on the specific biological response or mechanistic change, it was bryostatin-like, phorbol ester-like, intermediate in its behavior, or more effective than either. The pattern of response, moreover, varied depending on the conditions. We conclude that the newly emerging bryostatin derivatives such as Merle 23 provide powerful tools to dissect subsets of bryostatin mechanism and response. [Copyright &y& Elsevier]