Stoichiometric binding of diacylglycerol to the phorbol ester receptor

König, Bernhard; DiNitto, Patricia A.; Blumberg, Peter M.

doi:10.1002/jcb.240290105

Cited by 50 publications

(32 citation statements)

References 22 publications

(3 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Calcium (0.1 mM) did not affect the phosphatidylserine requirement for [ 3 H]PDBu binding to ␤2-chimaerin (EC 50 % ϭ 12.4 Ϯ 0.9%, n ϭ 3). The potency of phosphatidylserine for reconstitution of [ 3 H]PDBu binding to PKC has been reported to be dependent on calcium concentrations (36). Likewise, we observed that the EC 50 % for PKC␣ was significantly higher in the absence of calcium (40.0 Ϯ 1.4%, n ϭ 3) than in the presence of calcium (EC 50 %ϭ 1.5 Ϯ 0.1%, n ϭ 3).…”

Section: ␤2-chimaerin As a Phorbol Ester Receptorsupporting

confidence: 67%

“…A thorough mutagenesis analysis at 25 positions of the second cysteine-rich domain of PKC␦ has revealed essential amino acids involved in ligand recognition. Those residues are highly conserved in the cysteine-rich domain of ␤-chimaerins, including the conserved cysteines and histidines, conserved hydrophobic amino acids (positions 3,8,20,21,22,24,36,38, and 46), proline 11, and glutamine 27 (13). Several of those amino acids are missing or not conserved in cysteine-rich domains unable to bind phorbol esters, including those of atypical PKC , c-Raf, and Vav (23).…”

Section: Discussionmentioning

confidence: 99%

“…(35). Phosphatidylserine is the most effective phospholipid for supporting phorbol ester/DAG binding to PKC, n-chimaerin, and Unc-13 (26,30,36). Specific [ 3 H]PDBu binding to ␤2-chimaerin was dependent on the concentration of phosphatidylserine used in the assay (Fig.…”

Section: ␤2-chimaerin As a Phorbol Ester Receptormentioning

confidence: 93%

See 2 more Smart Citations

β2-Chimaerin Is a High Affinity Receptor for the Phorbol Ester Tumor Promoters

Caloca

Fernández

Lewin

et al. 1997

Journal of Biological Chemistry

Self Cite

113

View full text Add to dashboard Cite

␤2-chimaerin, a member of the GTPase-activating proteins for the small GTP-binding protein p21Rac, possesses a single cysteine-rich domain with high homology to those implicated in phorbol ester and diacylglycerol binding in protein kinase C (PKC) isozymes. We have expressed ␤2-chimaerin in Sf9 insect cells using the baculovirus expression system and determined that, like PKCs, ␤2-chimaerin binds phorbol esters with high affinity in the presence of phosphatidylserine as a cofactor. Scatchard plot analysis using the radioligand [ 3 H]phorbol 12,13-dibutyrate revealed a dissociation constant of 1.9 ؎ 0.2 nM for ␤2-chimaerin. Likewise, ␤2-chimaerin is a high affinity receptor for the bryostatins, a class of atypical PKC activators. A detailed comparison of structure-activity relations using several phorbol ester analogs revealed striking differences in binding recognition between ␤2-chimaerin and PKC␣. Although the diacylglycerol 1-oleoyl-2-acetylglycerol binds with similar potency to both ␤2-chimaerin and PKC␣, the mezerein analog thymeleatoxin has 56-fold less affinity for binding to ␤2-chimaerin. To establish whether ␤2-chimaerin responds to phorbol esters in cellular systems, we overexpressed ␤2-chimaerin in COS-7 cells and monitored its subcellular distribution after phorbol ester treatment. Interestingly, as described previously for PKC isozymes, ␤2-chimaerin translocates from cytosolic to particulate fractions as a consequence of phorbol ester treatment. Our results demonstrate that ␤2-chimaerin is a novel target for the phorbol ester tumor promoters. The expansion of the family of phorbol ester receptors strongly suggests a potential for the "nonkinase" receptors as cellular mediators of the phorbol ester responses.The phorbol esters and related diterpenes are natural compounds that are used widely as tumor promoters in animal models. The search for receptors for the phorbol esters led to the identification of protein kinase C (PKC) 1 as their target (1-5). The complexity and heterogeneity in the biology of phorbol esters suggested the existence of multiple receptors, and 11 PKC isoforms have been identified so far: classic or calciumdependent isozymes (PKC ␣, ␤1, ␤2, and ␥), novel or calciumindependent isozymes (PKC ␦, ⑀, , , and ), and atypical isozymes (PKC and ) (6, 7). The last group is unresponsive to the phorbol esters. DAG, the postulated endogenous activator of PKC, competes with phorbol esters for binding to PKC in in vitro assays, although its binding potency is lower than that of the most commonly used phorbol esters (8). The cysteine-rich domains present at the NH 2 -terminal region of the PKCs are the binding sites for DAG/phorbol esters (9 -13). Each of these 50-or 51-amino acid domains possesses the motif HX 12 CX 2 CX 13/14 CX 2 CX 4 HX 2 CX 7 C, where H is histidine, C is cysteine, and X is any other amino acid. This motif is duplicated in tandem in both the classic and novel PKCs and is present only once in the atypical PKCs. The cysteine-rich domains have been implicated in the associati...

show abstract

Section: ␤2-chimaerin As a Phorbol Ester Receptorsupporting

confidence: 67%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

β2-Chimaerin Is a High Affinity Receptor for the Phorbol Ester Tumor Promoters

Caloca

Fernández

Lewin

et al. 1997

Journal of Biological Chemistry

Self Cite

113

View full text Add to dashboard Cite

show abstract

“…1). Although phorbol esters bind to a similar site on PKC as does OlezGro [15], they do not produce the same effect on the Ca"' dependence of enzyme activity. Ole,Gro switches the CaZ + dependence from stimulatory to inhibitory, while with phorbol-12,13-dibutyrate, CaZC is still stimulatory (Fig.…”

Section: Discussionmentioning

confidence: 87%

Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA

Epand

Stafford

Lester

1992

European Journal of Biochemistry

View full text Add to dashboard Cite

Maximal protein kinase C activity with vesicles of phosphatidic acid and 1,2-di0leoyl-~sn-glycerol is observed in the absence of added Ca2 '. Addition of phosphatidylcholine to these vesicles restores some calcium dependence of enzyme activity. 1,2-Dioleoyl-sn-glycerol eliminates the Ca2 +-dependence of protein kinase C activity found with phosphatidic acid alone. Phorbol esters do not mimic the action of 1,2-dioleoyl-sn-glyceroI in this respect. This suggests that the 1,2-dioleoyl-sn-glycerol effect is a result of changes it causes in the physical properties of the membrane rather than to specific binding to the enzyme. The effect of 1,2-dioleoyl-.~n-glycerol on the phosphatidic-acid-stimulated protein kinase C activity is dependent on the molar Craction of 1,2-dioleoyl-sn-glycerol used and results in a gradual shift from Ca2+ stimulation at low 1,2-dioleoyl-sn-glycerol concentrations to calcium inhibition at higher concentrations of 1,2-dioleoyl-sn-glycerol. Phosphatidylserine-stimulated activity is also shown to be largely independent of the calcium concentration at higher molar fractions of 1,2-dioleoyl-sn-glycerol. Thus, with certain lipid compositions, protein kinase C activity becomes independent of the calcium concentration or requires only very low, stoichiometric binding of Ca2 + to high affinity sites on the enzyme.Protein kinase C can bind to phosphatidic acid vesicles more readily than it can bind to phosphatidylserine vesicles in the absence of calcium. Addition of 1,2-dioleoyl-sn-glycerol to phosphatidylserine vesicles promotes the partitioning of protein kinase C into the membrane in the absence of added Ca2 ' . There is no isozyme specificity in this binding. These results suggest that a less-tightly packed headgroup region of the bilayer causes increased insertion of protein kinase C into the membrane. This is a necessary but not sufficient condition for activation of the enzyme in the presence of EGTA.Protein kinase C (PKC) plays an important role in cell regulation and is the receptor for the phorbol ester tumor promoters. This protein kinase was first identified as a calcium-dependent and phospholipid-dependent enzyme [ l]. The phospholipid requirement for the enzyme is specific for anionic lipids with phosphatidylserine being most effective [2] but phosphatidic acid [2 -41, cardiolipin [3] and phosphatidylinositol [2] are also active. Calcium binds to PKC only in the presence of phospholipid [5] and the binding affinity for Ca2+ is increased with more phosphatidylserine in the membrane [6]. Diacylglycerols markedly increase the sensitivity of PKC to activation by Ca2+ in the presence of phospholipid [7]. In contrast to phosphatidylserine, we find that addition of 1,2-dioleoyl-sn-glycerol (Ole,Gro) to phosphatidic acid causes a calcium-induced inhibition, rather than activation, of the Correspondence to

show abstract

“…The C1 domain stereospecifically binds the anionic phospholipid phosphatidylserine (46,70). Pioneering work by Blumberg and colleagues, who developed lipophilic analogs of phorbol esters that allowed, for the first time, demonstration of specific and saturable binding of phorbol esters to cells (22), identified the stoichiometry of phorbol ester binding as 1 mol ligand/mol PKC (53); for most isozymes, it is the C1b domain that is the relevant binding module in the context of the full-length protein (49,80). The C2 domain of conventional PKC isozymes binds anionic phospholipids with modest, but not stereospecific, selectivity for phosphatidylserine (16,19,46,61).…”

Section: The Pkc Familymentioning

confidence: 99%

Protein kinase C: poised to signal

Newton

2010

American Journal of Physiology-Endocrinology and Metabolism

463

509

View full text Add to dashboard Cite

Nestled at the tip of a branch of the kinome, protein kinase C (PKC) family members are poised to transduce signals emanating from the cell surface. Cell membranes provide the platform for PKC function, supporting the maturation of PKC through phosphorylation, its allosteric activation by binding specific lipids, and, ultimately, promoting the downregulation of the enzyme. These regulatory mechanisms precisely control the level of signalingcompetent PKC in the cell. Disruption of this regulation results in pathophysiological states, most notably cancer, where PKC levels are often grossly altered. This review introduces the PKC family and then focuses on recent advances in understanding the cellular regulation of its diacylglycerol-regulated members.calcium; diacylglycerol; phorbol esters THE FAMILY OF PKC ISOZYMES transduces the myriad of signals resulting from receptor-mediated hydrolysis of phospholipids, playing critical roles in diverse cellular functions. The discovery in the 1980s that they are the receptors for the potent tumor promoting phorbol esters, coupled with the discovery that they mediate signaling by the lipid second messenger diacylglycerol, secured a center stage position for this family of enzymes in cellular regulation (6, 66). Family members, themselves, are regulated by precise mechanisms that control their structure, function, and subcellular localization. This review discusses our current understanding of the mechanisms controlling protein kinase C signaling. The PKC FamilyThe PKC family occupies the tip of a branch of the AGC kinases from which the related kinases protein kinase N (PKN), Akt/PKB, p70 S6 kinase, and the phosphoinositidedependent kinase-1 (PDK-1) diverge (Fig. 1A). The 10 members that populate the mammalian family evolved from the single PKC in saccharomyces cerivisiae, PKC1 (57, 98), and are grouped into three classes based on their domain composition (Fig. 1B) (71). This domain composition, in turn, dictates the cofactor regulation of the classes (Fig. 1C). At the very tip of the branch are the four conventional isozymes: PKC␣, the first isozyme cloned (76); the alternatively spliced PKC␤I and PKC␤II (which differ in the last 43 amino acids) and PKC␥; next are the four novel isozymes, PKC␦, -ε, -, and -; closest to the point of divergence are the atypical isozymes PKC and PKC (human; murine isozyme is PKC).All family members share the same architecture: a carboxylterminal kinase domain linked by a flexible hinge segment to an amino-terminal region containing regulatory modules (Fig. 1B) (69, 77). These regulatory modules confer sensitivity to the second messengers diacylglycerol (C1 domain) or Ca 2ϩ (C2 domain), although, importantly, some isozymes have variants of these modules that do not bind ligand (8,20).Conventional isozymes contain tandem C1 domains that bind diacylglycerol and a C2 domain that binds anionic lipids in a Ca 2ϩ -dependent manner. The small globular C1 domain is also the binding site for the potent tumor-promoting phorbol esters (43, 100), which bi...

show abstract

Stoichiometric binding of diacylglycerol to the phorbol ester receptor

Cited by 50 publications

References 22 publications

β2-Chimaerin Is a High Affinity Receptor for the Phorbol Ester Tumor Promoters

β2-Chimaerin Is a High Affinity Receptor for the Phorbol Ester Tumor Promoters

Lipid vesicles which can bind to protein kinase C and activate the enzyme in the presence of EGTA

Protein kinase C: poised to signal

Contact Info

Product

Resources

About