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Retinoids as ligands and coactivators of protein kinase C alpha¹

Program in Immunology, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA

SPECIFIC AIM

Vitamin A and carotenes, a family of molecules dating back in evolution more than 500 million years, perform a plethora of tasks in nearly all genera. The individual family members, such as retinaldehyde and carotenes, are engaged in photosynthetic systems of bacteria and plants, respectively; retinaldehydes form the universal chemical engine of vision; retinoic acids help regulate transcription in vertebrates; and, according to studies presented in this report, hydroxylated vitamin A metabolites serve as regulators of redox signaling pathways.

Immunologists are aware of the negative effects of vitamin A deficiency on human health. While studying an in vitro model of vitamin A deficiency, we discovered a decade ago a new class of hydroxylated retinoids that could potentially serve as the biological mediators of vitamin A in the immune system. Lymphocytes are prone to apoptosis in situations of vitamin A deprivation. The outstanding property of hydroxylated retinoids was their ability to replace retinol, promote cell survival, and forestall apoptosis, something that retinoic acid (RA) was not able to do. Therefore, this new class holds much promise for solving an old mystery.

Meanwhile, it is becoming apparent that the function of hydroxylated retinoids is much broader. Nearly all cells in the body have the ability to convert vitamin A to 14-hydroxy-retro-retinol (HRR) and 13,14 di-hydroxy-retinol; because this biochemical capacity is conserved from insects to humans, we attach a vital role to these vitamin A metabolites that might well affect a large number of tissues. The receptors of hydroxylated retinoids were recently identified in our laboratory as members of the serine/threonine kinase family, cRaf and protein kinase C (PKC). They all bind retinoids with nanomolar affinity. The binding sites have been mapped to the cysteine-rich domains, the conserved zinc fingers these kinases share in common. As many more cytoplasmic proteins harbor zinc fingers, the family of retinol binding proteins is likely to grow in the future.

The aim of the present study was to establish this new paradigm for one PKC isoform, alpha. The capacity of a variety of retinoids to bind the cysteine-rich domains of PKC was determined and the functional consequences of binding hydroxylated retinoids to the cysteine-rich domain were explored, with special emphasis on the question of whether the retinoids tested exert differential effects on this enzyme. The surprising answer was obtained that they function in redox regulation of PKC and that different retinoids indeed produced different effects.

PRINCIPAL FINDINGS

Four different fluorometric methods were applied to determine the binding affinities of the retinoids: retinol, HRR, anhydroretinol (AR), and RA. The cysteine-rich domains of PKC, , and µ (each containing two tandemly arranged homologues denoted C1A and C1B) were tested as Gst fusion proteins for their capacity to bind retinoids. Quenching of the intrinsic protein fluorescence by all four retinoids indicated binding. In the example of retinol, a second fluorometric method showed the emergence of a fluorescence energy transfer signal, confirming that binding to the PKC C1A domain had occurred (see Fig. 1 ). Titrations of retinoids led to saturation curves, and curve fitting by the theorem of Norris et al. furnished the apparent dissociation constants. All four retinoids bound with similar affinities in the nanomolar range. Both the C1A and C1B domains of the three isoforms tested gave evidence of binding of four retinoids with comparable affinities. The exception was PKC C1B, which did not bind any of the four despite close sequence homology to those isoforms that bound. Binding was confirmed by a third method, fluorescence enhancement of the bound ligand. Titrations of retinoids by this method indicated saturable binding, and computations of apparent dissociation constants yielded values in close agreement with those obtained by the quench method. As binding of retinol produced red shift and vibronic fine structure in the fluorescence excitation spectrum, a fourth independent method signaled binding.

View larger version (20K): [in this window] [in a new window]   Figure 1. Binding of retinol to the cysteine-rich domain of PKC. The intrinsic protein fluorescence of the Gst fusion protein containing the C1A domain of PKC is shown to suffer quenching upon addition of equimolar amount of retinol, indicating binding. Fluorescence resonance energy transfer to retinol (insert) confirmed binding. The corresponding C1B domain showed neither quenching nor FRET, and hence does not bind retinol. Titrations of retinol yielded saturable quench curves from which the apparent dissociation constants were computed. Other retinoids also produced quenching, but no FRET signal.

The cysteine-rich domains of PKC harbor the binding sites of known activators of PKC: diglycerides and phorbol ester. Using three independent binding competition methods, the retinoid binding sites were shown to be distinct from those of the former.

Unlike other lipids such as diglycerides and phorbol esters, which bind the same zinc finger domains (albeit at a separate site) and activate PKC family members, retinoids on their own did not activate serine/threonine kinases. In combination with reactive oxygen species, however, retinol and HRR significantly enhanced the activation of PKC (Fig. 2 ). We surmise, therefore, that hydroxylated retinoids, by binding the zinc finger domain, allow the efficient and selective oxidation of critical cysteine residues. However, the fact that neither AR nor RA produced appreciable enhancement of redox activation suggested differential action among different retinoids.

View larger version (22K): [in this window] [in a new window]   Figure 2. Enhancement of redox activation of PKC by retinol. 3T3 cells were activated by hydrogen peroxide (100 µM) in the presence or absence of retinol. PKC kinase activity was determined in immunoprecipitates over the time course shown. A) A physiological concentration of retinol greatly enhanced the PKC activity (e.g. on average 2.8-fold at 10 min time point; P > 0.002; n=6). B) HRR at 10-7 M produced marked enhancement of PKC activation in the redox pathway (a threefold difference at 100 µM peroxide; P > 0.001; n=6).

CONCLUSIONS AND SIGNIFICANCE

The unprecedented result was obtained that retinoids bound the cysteine-rich domains of several PKC isoforms in vitro with nanomolar affinities. Although formal proof is still outstanding on whether they would do so in vivo as well, this appears highly likely, since phorbol ester and diglycerides bind instantaneously in vivo to the same domain. Therefore, the cysteine-rich domains are accessible to small lipids, such as retinol. As it is abundant in plasma and can pass freely into cells, retinol or its metabolites are likely to occupy all sites on PKC constitutively.

Next to binding, the second outstanding result was that retinol and HRR markedly increased the nonclassical activation of PKC by reactive oxygen species. While it is unclear how retinoids can enhance the activation process, they seem to act in a manner of catalysts, since they facilitate the oxidation process. This link to redox regulation is not surprising, as these compounds have long been known to possess both pro- and antioxidant properties, the former dominating in this case. Our results may speak to the unsolved problem in redox regulation as to how specific cysteine residues in proteins, which harbor many, can be selectively oxidized. The association of the pro-oxidant retinoid with structures rich in cysteines is hardly accidental and conveys a message. It suggests that the local, catalyst-like action of the bound retinoid promotes the targeted oxidation of select cysteines (see Fig. 3 ). One manner in which such preferential oxidation could occur is through facilitation of electron transfer. Another way might be an increase in the electronegativity of thiol groups in the immediate vicinity of bound retinoid, although no precedent for such electrochemical action is known. It is conceivable that individual retinoids differ in the degree to which they impart electrochemical changes to the protein, explaining the observed differences of redox enhancement.

View larger version (42K): [in this window] [in a new window]   Figure 3. Heuristic schematic diagram depicting the action of retinoids in redox activation of PKC. The PKC molecule is shown in its accepted domain structure (from amino to carboxyl terminus: pseudosubstrate domain as yellow circle; cysteine-rich domains C1A and C1B in magenta; the C2 and catalytic domains in plum). Reactive oxygen is proposed to oxidize cysteines in cysteine-rich domains, compromising the ability to chelate zinc and causing the zinc finger to open (X and box-like symbols in green represent intact and oxidation-compromised zinc coordination centers, respectively). Similar to activation by lipid agonists, an initial conformational change in the cysteine-rich domains allows the molecule to unfold and translocate to the cell membrane. This topological change is thought to be complemented by secondary phosphorylations, Ca and phosphatidylserine binding, to lock the catalytic domain (cat) into an active state. Binding of retinol or HRR to C1A may change the local electrochemical properties (indicated by shading in pink), thereby targeting select cysteine residues for preferential oxidation and initiating high kinase activity (long arrows). Oxidation can also take place in the absence of retinol, but activation would proceed with less efficiency (short arrows).

Like the homologous structures of certain bacterial enzymes, the zinc finger domains of mammalian serine/threonine kinases could function as reversible redox switches. Indeed our finding that changing experimentally the microenvironment from oxidant to reducing caused the prompt inactivation of PKC supports such a notion. The retinoids could act as devices to assure that chemical changes are directed to those cysteine residues that need to be modified as opposed to those that need to be spared. The observation of differential action raised for the different retinoids suggests an opportunity for fine-tuning the redox switch. Taken together, our results open a new field of study for vitamin A, placing this interesting molecule at the crossroads of signal transduction and redox regulation.

UNRESOLVED ISSUES

No information to date is available on the chemistry of redox activation of PKC, whether one or more Zn chelating centers are targeted. PKC contains a total of four—one pair in C1A and another in C1B. Whether one or more of theses centers or precisely which cysteines are affected, or whether oxidation of thiols would lead to the release of Zn²⁺ with consequent conformational change, as might be predicted from the elegant work on the bacterial heat shock protein Hsp33, is yet to be investigated. The beauty of the idea is that transfer of a mere two electrons could initiate vast topological change in a readily reversible manner. Signaling requires such simple and reversible switches. To understand the role of retinoids for redox regulation of PKC, much structural and biochemical work needs to be done with simple model peptides, including crystallography and nuclear magnetic resonance. Ultimately, the answer to how retinoids work may lie in the realm of protein electrochemistry.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0329fje To cite this article, use (November 14, 2000) FASEB J. 10.1096/fj.00-0329fje

² A.I., B.H., and C.S. contributed equally to this work.

³ Correspondence: Immunology Program, Memorial Sloan Kettering Cancer Center, 1275 York Ave., New York, NY 10021, USA. E-mail: u-hammerling{at}ski.mskcc.org

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