Victor M. Samokyszyn

Direct Interaction of All-trans-retinoic Acid with Protein Kinase C (PKC) IMPLICATIONS FOR PKC SIGNALING AND CANCER THERAPY

Protein kinase C (PKC) regulates fundamental cellular functions including proliferation, differentiation, tumorigenesis, and apoptosis. All-trans-retinoic acid (atRA) modulates PKC activity, but the mechanism of this regulation is unknown. Amino acid alignments and crystal structure analysis of retinoic acid (RA)-binding proteins revealed a putative atRA-binding motif in PKC, suggesting existence of an atRA binding site on the PKC molecule. This was supported by photolabeling studies showing concentration- and UV-dependent photoincorporation of [3H]atRA into PKCα, which was effectively protected by 4-OH-atRA, 9-cis-RA, and atRA glucuronide, but not by retinol. Photoaffinity labeling demonstrated strong competition between atRA and phosphatidylserine (PS) for binding to PKCα, a slight competition with phorbol-12-myristate-13-acetate, and none with diacylglycerol, fatty acids, or Ca2+. At pharmacological concentrations (10 μm), atRA decreased PKCα activity through the competition with PS but not phorbol-12-myristate-13-acetate, diacylglycerol, or Ca2+. These results let us hypothesize that in vivo, pharmacological concentrations of atRA may hamper binding of PS to PKCα and prevent PKCα activation. Thus, this study provides the first evidence for direct binding of atRA to PKC isozymes and suggests the existence of a general mechanism for regulation of PKC activity during exposure to retinoids, as in retinoid-based cancer therapy.

Application of photoaffinity labeling with [3H] all trans- and 9-cis-retinoic acids for characterization of cellular retinoic acid–binding proteins I and II

Cellular retinoic acid–binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all‐trans‐retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity‐based binding assay involving competition between potential ligands of CRABP and [3H]atRA or [3H]‐9‐cis‐RA for binding to the atRA‐binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [3H]atRA was light‐ and concentration‐dependent, saturable, and protected by several retinoids in a concentration‐dependent manner, indicating that binding occurred in the CRABP atRA‐binding site. Structure–function relationship studies demonstrated that oxidative changes to the atRA β‐ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6‐epoxy‐RA and retinoyl‐β‐D‐glucuronide (RAG). The labeling of both CRABPs with 9‐cis‐RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP‐glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [3H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA‐binding site of these proteins.

A theoretical investigation of endocyclic allylic carbon-centered radical formation in retinoic acid

Abstract We have carried out theoretical calculations that suggest that C4 carbon-centered radical formation in ( E )- and (13 Z )-retinoic acid, catalyzed by cytochromes P450 and PGH synthase, occur by a direct H-atom abstraction mechanism rather than an electron abstraction mechanism. In addition, our calculations indicate that C4 radical formation in the 13 Z -isomer is thermochemically preferred compared with the all trans -isomer.