Rapid Assay of Binding of Tumor-Promoting Phorbol Esters to Protein Kinase C1

The family of homologous enzymes known as protein kinase C (PKC) has been the object of intense interest because of its crucial role in cellular signal transduction. Although considerable information about the activation of PKC has been gained through structure-activity, molecular modeling, and synthetic studies of both natural and designed activators, information about the structure of PKC itself has been limited by its large Since the identification of its pivotal role in cellular signal transduction (1), the family of enzymes known as protein kinase C (PKC) has served as a key lead in the elucidation of normal and abnormal cellular processes. Under normal circumstances, PKC is activated by the binding of 1,2-diacyl-snglycerol (DAG, structure 1) (2-4), a second messenger released in response to the binding of extracellular agents to cell surface receptors. Differing significantly in structure from DAG, plant-derived phorbol esters (structure 2) and a diverse range of other agents (5) also bind to and activate PKC in competition with DAG but, unlike DAG, initiate a series of abnormal events associated with tumor promotion. In contrast, prostratin (structure 3), which differs from the phorbol esters only as a result of an ester deletion at the C-12 position, is not a potent promoter but exhibits cytoprotective effects in cells infected with the human immunodeficiency virus (6), a finding of potential consequence in AIDS therapy. Representative of a third structural class of activators, the marinederived macrocyclic lactone bryostatin (structure 4), now in clinical trials, binds to PKC in competition with phorbol esters but blocks a subset of phorbol ester-induced responses includ-

Section: Methodssupporting

confidence: 84%

mentioning

confidence: 99%

Identification, activity, and structural studies of peptides incorporating the phorbol ester-binding domain of protein kinase C.

Wender

Irie

Miller

1995

“…Therefore, specific binding sites for PBt2 are concentrated in the stratum radiatum (the region containing the CA1 apical dendrites). Binding assays using 30 nM [3H]PBt2 (12) revealed that in naive animals, the PKC distribution is virtually identical to that obtained in naive and pseudoconditioned animals by enzymatic activity assay of PKC purified from cytosolic and membrane fractions. Therefore, the enzyme assay appears to provide a valid indication of PKC distribution.…”

Section: Methodsmentioning

confidence: 57%

Classical conditioning induces long-term translocation of protein kinase C in rabbit hippocampal CA1 cells.

Bank

Deweer

Kuzirian

et al. 1988

139

The role of the Ca2 /phospholipid-dependent, diacylglycerol-activated enzyme protein kinase C (PKC) in rabbit eyelid conditioning was examined. PKC was partially purified from the CAl region of hippocampal slices from naive, pseudoconditioned, and conditioned rabbits 24 hr after the rabbits were well conditioned. Crude membrane and cytosol fractions were prepared. In conditioned rabbits, significantly more PKC activity (63.3%) was associated with the membrane fraction (and significantly less with the cytosol fraction) compared to naive (42.0%) and pseudoconditioned (44.7%) animals. These differences in distribution of enzyme activity were paralleled by differences in stimulation of enzyme activity by Ca2", phospholipid, and diacylglycerol.There were no between-group differences in basal protein kinase activity. These results suggest that there is a long-term translocation of PKC from cytosol to membrane as a result of conditioning. Autoradiographic binding of radioactive phorbol 12,13-dibutyrate to PKC demonstrated that almost all specific binding was in the stratum radiatum, a region containing the proximal apical dendrites of CAl pyramidal neurons. Therefore, this may be the site of the conditioningspecific PKC translocation, a locus well-suited to underlie the biophysical effects of conditioning.Recent work in our laboratory has established that classical conditioning of the nictitating membrane/eyelid response causes a biophysical record similar to that of Hermissenda classical conditioning-both records involving persistent reductions of a Ca2+-dependent outward K+ current, IK(Ca)(1-4). A long-lasting decrease in IK(Ca) was measured as a decrease in the late afterhyperpolarization (AHP) following impulses of hippocampal CA1 cells (1, 2). In addition, it was found that conditioning induces an enhancement in the summation of postsynaptic potentials (5). Both the AHP reduction and the enhanced postsynaptic potential summation can be mimicked by bath application of phorbol esters, a class of pharmacological agents that activate Ca2+/phospholipid-dependent protein kinase C (PKC) (5-7). Similarly, in Hermissenda, injection of purified PKC into type B photoreceptors reduces IA (an early, rapidly inactivated outward K+ current) and IK(Ca)' but only when paired with a "calcium load" (8). We therefore examined the role of PKC activation as a possible biochemical mechanism underlying the biophysical changes induced by classical conditioning. We now report a molecular record of nictitating membrane/eyelid conditioning: a long-lasting (24 hr), conditioning-specific translocation of PKC activity from the cytosol to the membrane compartment of CA1 cells isolated from the hippocampal slice. This finding is novel because the translocation is of long duration, is learning-specific, and is intrinsic to a population of vertebrate neurons known to undergo biophysical alterations as a result of this type of learning. In addition, the present finding provides a possible point of convergence between mechanisms of learning a...

“…The vials were incubated at 37°C for 100 min and then placed on ice for 5 min. The samples were filtered and washed as previously described (23). The filters were soaked for 12 hr in 3 ml of CytoScint scintillation fluid before counting.…”

Section: Methodsmentioning

confidence: 99%

The design, computer modeling, solution structure, and biological evaluation of synthetic analogs of bryostatin 1

Wender

Debrabander

Harran

et al. 1998

107

The bryostatins are a unique family of emerging cancer chemotherapeutic candidates isolated from marine bryozoa. Although the biochemical basis for their therapeutic activity is not known, these macrolactones exhibit high affinities for protein kinase C (PKC) isozymes, compete for the phorbol ester binding site on PKC, and stimulate kinase activity in vitro and in vivo. Unlike the phorbol esters, they are not first-stage tumor promoters. The design, computer modeling, NMR solution structure, PKC binding, and functional assays of a unique class of synthetic bryostatin analogs are described. These analogs (7b, 7c, and 8) retain the putative recognition domain of the bryostatins but are simplified through deletions and modifications in the C4-C14 spacer domain. Computer modeling of an analog prototype (7a) indicates that it exists preferentially in two distinct conformational classes, one in close agreement with the crystal structure of bryostatin 1. The solution structure of synthetic analog 7c was determined by NMR spectroscopy and found to be very similar to the previously reported structures of bryostatins 1 and 10. Analogs 7b, 7c, and 8 bound strongly to PKC isozymes with K i ‫؍‬ 297, 3.4, and 8.3 nM, respectively. Control 7d, like the corresponding bryostatin derivative, exhibited weak PKC affinity, as did the derivative, 9, lacking the spacer domain. Like bryostatin, acetal 7c exhibited significant levels of in vitro growth inhibitory activity (1.8-170 ng͞ml) against several human cancer cell lines, providing an important step toward the development of simplified, synthetically accessible analogs of the bryostatins.