The PKD (protein kinase D) family are novel DAG (diacylglycerol) receptors. The twin C1 domains of PKD, designated C1a and C1b, have been shown to bind DAG or phorbol esters. However, their ligand-binding activities and selectivities have not been fully characterized. Here, binding activities of isolated C1a, C1b and intact C1a–C1b domains to DAG and phorbol esters were analysed. The isolated C1b domains of PKD isoforms bind [3H]PDBu ([20-3H]phorbol 12, 13-dibutyrate) with similar high affinities, while they exhibit weaker affinities towards a synthetic DAG analogue, DOG (1,2-dioctanoyl-sn-glycerol), as compared to the control. Mutating a conserved lysine residue at position 22 to tryptophan in C1b of PKD3 fully restores its affinity to DOG, indicating that this residue accounts for its weaker affinity to DOG. In contrast, the non-consensus residues in the isolated C1a domain of PKD mainly contribute to maintaining the protein's structural fold, since converting these residues in C1a of PKD3 to those in PKD1 or PKD2 drastically reduces the maximal number of active receptors, while only minimally impacting ligand-binding activities. Moreover, ligand-binding activities of C1a and C1b are sensitive to the structural context in an intact C1a–C1b domain and exhibit unique patterns of ligand selectivity. C1a and C1b in the intact C1a–C1b of PKD1 are opposite in selectivity for PDBu and DOG. In contrast, C1a of PKD3 exhibits 48-fold higher affinity to DOG as compared to C1b, although both domains bind PDBu with equivalent affinities. Accordingly, mutating C1a of a full-length PKD3–GFP greatly reduces DOG-induced plasma membrane translocation, but does not affect that induced by PMA. In summary, individual C1 domains of PKD isoforms differ in ligand-binding activity and selectivity, implying isoform-selective regulation of PKD by phorbol esters and DAG.
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Research Article|
March 27 2008
Selective binding of phorbol esters and diacylglycerol by individual C1 domains of the PKD family
Jun Chen;
Jun Chen
1Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
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Fan Deng;
Fan Deng
1Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
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Jun Li;
Jun Li
1Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
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Q. Jane Wang
Q. Jane Wang
1
1Department of Pharmacology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A.
1To whom correspondence should be addressed (email qjw1@pitt.edu).
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Publisher: Portland Press Ltd
Received:
September 27 2007
Revision Received:
December 12 2007
Accepted:
December 13 2007
Accepted Manuscript online:
December 13 2007
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© The Authors Journal compilation © 2008 Biochemical Society
2008
Biochem J (2008) 411 (2): 333–342.
Article history
Received:
September 27 2007
Revision Received:
December 12 2007
Accepted:
December 13 2007
Accepted Manuscript online:
December 13 2007
Citation
Jun Chen, Fan Deng, Jun Li, Q. Jane Wang; Selective binding of phorbol esters and diacylglycerol by individual C1 domains of the PKD family. Biochem J 15 April 2008; 411 (2): 333–342. doi: https://doi.org/10.1042/BJ20071334
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