Residues in the Second Cysteine-rich Region of Protein Kinase C δ Relevant to Phorbol Ester Binding as Revealed by Site-directed Mutagenesis

Kazanietz, Marcelo G.; Wang, Shaomeng; Milne, G. W. A.; Lewin, Nancy E.; Liu, Howard L.; Blumberg, Peter M.

doi:10.1074/jbc.270.37.21852

Cited by 158 publications

(191 citation statements)

References 30 publications

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“…We carefully analyzed the cysteine-rich sequences of the C1 domains of all DGK isozymes on the basis of the 20 residues (Fig. 2) critical to PDBu binding (16,24,32). DGK␤-C1A, DGK␥-C1A, DGK␦-C1A, DGK␦-C1B, DGK -C1A, and DGK -C1C have the same core structure as the PKC C1 domains, where six cysteines and two histidines are conserved in the pattern HX 12 CX 2 CX 13-14 CX 2 CX 4 HX 2 CX 7 C. Table II summarizes the percentages of sequence homology of all DGK C1 domains to the 20 residues.…”

Section: Resultsmentioning

confidence: 99%

Synthesis and Phorbol Ester Binding of the Cysteine-rich Domains of Diacylglycerol Kinase (DGK) Isozymes

Shindo¹,

Irie

Masuda

et al. 2003

Journal of Biological Chemistry

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Diacylglycerol kinase (DGK) and protein kinase C (PKC) are two distinct enzyme families associated with diacylglycerol. Both enzymes have cysteine-rich C1 domains (C1A, C1B, and C1C) in the regulatory region. Although most PKC C1 domains strongly bind phorbol esters, there has been no direct evidence that DGK C1 domains bind phorbol esters. We synthesized 11 cysteine-rich sequences of DGK C1 domains with good sequence homology to those of the PKC C1 domains. Among them, only DGK␥-C1A and DGK␤-C1A exhibited significant binding to phorbol 12,13-dibutyrate (PDBu). Scatchard analysis of rat-DGK␥-C1A, human-DGK␥-C1A, and human-DGK␤-C1A gave K d values of 3.6, 2.8, and 14.6 nM, respectively, suggesting that DGK␥ and DGK␤ are new targets of phorbol esters. An A12T mutation of human-DGK␤-C1A enhanced the affinity to bind PDBu, indicating that the ␤-hydroxyl group of Thr-12 significantly contributes to the binding. The K d value for PDBu of FLAG-tagged whole rat-DGK␥ (4.4 nM) was nearly equal to that of rat-DGK␥-C1A (3.6 nM). Moreover, 12-O-tetradecanoylphorbol 13-acetate induced the irreversible translocation of whole rat-DGK␥ and its C1B deletion mutant, not the C1A deletion mutant, from the cytoplasm to the plasma membrane of CHO-K1 cells. These results indicate that 12-O-tetradecanoylphorbol 13-acetate binds to C1A of DGK␥ to cause its translocation.Diacylglycerol kinase (DGK) 1 and protein kinase C (PKC) both interact with the second messenger diacylglycerol (DG) (1, 2). DGK phosphorylates DG to produce phosphatidic acid, whereas PKC is allosterically activated by DG in the presence of phosphatidylserine. Therefore, DGK may inhibit the activation of PKC by attenuating DG levels, contributing to the regulation of intracellular signal transduction.To date, nine subtypes of mammalian DGKs have been cloned (3-15). All DGK isozymes consist of a conserved catalytic domain and two or three cysteine-rich C1 domains designated as C1A, C1B, and C1C (16). These isozymes are classified into five classes according to the other functional domains (Fig. 1). The class I isozymes (DGK␣, -␤, and -␥) have calcium binding domains (EF-hands). The class II isozymes (DGK␦ and -) have a pleckstrin homology domain at the N terminus, and their catalytic region is split into two domains unlike the other DGK isozymes. DGK⑀ has a simple structure and is classified as a class III isozyme. The class IV isozymes DGK and have a myristoylated alanine-rich C kinase substrate homology domain and four ankyrin repeats. DGK , which has three C1 domains unlike other DGK and PKC isozymes, is the only isozyme in class V.The similarity between DGK and PKC isozymes in structure is in the cysteine-rich C1 domains. Recent investigations using NMR spectroscopy and x-ray crystallography have revealed the three-dimensional structure of C1B domains of PKC␣, PKC␥,. Each PKC C1 domain has six conserved cysteines and two histidines in the typical core structure HX 12 CX 2 CX 13-14 CX 2 CX 4 HX 2 CX 7 C (where X is any amino acid) that coordinates two atoms of zinc in...

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Section: Resultsmentioning

confidence: 99%

Synthesis and Phorbol Ester Binding of the Cysteine-rich Domains of Diacylglycerol Kinase (DGK) Isozymes

Shindo¹,

Irie

Masuda

et al. 2003

Journal of Biological Chemistry

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“…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%

“…Although the cysteine-rich domains of ␤-chimaerins have a high degree of homology to those in PKC isozymes and nchimaerin, it is not known whether the ␤-chimaerins are receptors for the phorbol esters and DAG. It is noteworthy that single changes at several positions of the cysteine-rich domains in PKCs are sufficient to abolish completely or reduce substantially the binding of phorbol esters (13). Moreover, not all of the proteins possessing similar cysteine-rich motifs in their structure are phorbol ester receptors (23,24).…”

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confidence: 99%

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

Caloca

Fernández

Lewin

et al. 1997

Journal of Biological Chemistry

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113

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␤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...

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“…C1 modules in c-Raf and PKC do not bind PMA or DAG). Mutation of a conserved Pro at position 11 in the C1 module provides an incisive test (41,50 Cells expressing wild type (WT) or mutant DKF-2 proteins were treated with vehicle or PMA for 10 min and then fractionated into cytosol (supernatant) and total membranes (pellet) (see "Experimental Procedures"). All cytosol samples had low basal (background) kinase activity that was not affected by PMA.…”

Section: Resultsmentioning

confidence: 99%