Activation Mechanisms of Conventional Protein Kinase C Isoforms Are Determined by the Ligand Affinity and Conformational Flexibility of Their C1 Domains

Bharath Ananthanarayanan, Robert Stahelin, Michelle A. Digman, Wonhwa Cho

Research output: Contribution to journalArticle

107 Citations (Scopus)

Abstract

The regulatory domains of conventional and novel protein kinases C (PKC) have two C1 domains (C1A and C1B) that have been identified as the interaction site for diacylglycerol (DAG) and phorbol ester. It has been reported that C1A and C1B domains of individual PKC isoforms play different roles in their membrane binding and activation; however, DAG affinity of individual C1 domains has not been quantitatively determined. In this study, we measured the affinity of isolated C1A and C1B domains of two conventional PKCs, PKCα and PKCγ, for soluble and membrane-incorporated DAG and phorbol ester by isothermal calorimetry and surface plasmon resonance. The C1A and C1B domains of PKCα have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. In contrast, the C1A and C1b domains of PKCγ have comparably high affinities for both DAG and phorbol ester. Consistent with these results, mutational studies of full-length proteins showed that the C1A domain is critical for the DAG-induced activation of PKCα, whereas both C1A and C1B domains are involved in the DAG-induced activation of PKCγ. Further mutational studies in conjunction with in vitro activity assay and monolayer penetration analysis indicated that, unlike the C1A domain of PKCα, neither the C1A nor the C1B domain of PKCγ is conformationally restricted. Cell studies with enhanced green fluorescent protein-tagged PKCs showed that PKCγ did not translocate to the plasma membrane in response to DAG at a basal intracellular calcium concentration due to the inaccessibility of its C1A domain, whereas PKCγ rapidly translocated to the plasma membrane under the same conditions. These data suggest that differential activation mechanisms of PKC isoforms are determined by the DAG affinity and conformational flexibility of their C1 domains.

Original languageEnglish (US)
Pages (from-to)46886-46894
Number of pages9
JournalJournal of Biological Chemistry
Volume278
Issue number47
DOIs
StatePublished - Nov 21 2003
Externally publishedYes

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Diglycerides
Protein Kinase C
Protein Isoforms
Chemical activation
Ligands
Phorbol Esters
Cell membranes
Cell Membrane
Membranes
Calorimetry
Surface Plasmon Resonance
Surface plasmon resonance
Monolayers
Assays
Calcium

ASJC Scopus subject areas

  • Biochemistry

Cite this

Activation Mechanisms of Conventional Protein Kinase C Isoforms Are Determined by the Ligand Affinity and Conformational Flexibility of Their C1 Domains. / Ananthanarayanan, Bharath; Stahelin, Robert; Digman, Michelle A.; Cho, Wonhwa.

In: Journal of Biological Chemistry, Vol. 278, No. 47, 21.11.2003, p. 46886-46894.

Research output: Contribution to journalArticle

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abstract = "The regulatory domains of conventional and novel protein kinases C (PKC) have two C1 domains (C1A and C1B) that have been identified as the interaction site for diacylglycerol (DAG) and phorbol ester. It has been reported that C1A and C1B domains of individual PKC isoforms play different roles in their membrane binding and activation; however, DAG affinity of individual C1 domains has not been quantitatively determined. In this study, we measured the affinity of isolated C1A and C1B domains of two conventional PKCs, PKCα and PKCγ, for soluble and membrane-incorporated DAG and phorbol ester by isothermal calorimetry and surface plasmon resonance. The C1A and C1B domains of PKCα have opposite affinities for DAG and phorbol ester; i.e. the C1A domain with high affinity for DAG and the C1B domain with high affinity for phorbol ester. In contrast, the C1A and C1b domains of PKCγ have comparably high affinities for both DAG and phorbol ester. Consistent with these results, mutational studies of full-length proteins showed that the C1A domain is critical for the DAG-induced activation of PKCα, whereas both C1A and C1B domains are involved in the DAG-induced activation of PKCγ. Further mutational studies in conjunction with in vitro activity assay and monolayer penetration analysis indicated that, unlike the C1A domain of PKCα, neither the C1A nor the C1B domain of PKCγ is conformationally restricted. Cell studies with enhanced green fluorescent protein-tagged PKCs showed that PKCγ did not translocate to the plasma membrane in response to DAG at a basal intracellular calcium concentration due to the inaccessibility of its C1A domain, whereas PKCγ rapidly translocated to the plasma membrane under the same conditions. These data suggest that differential activation mechanisms of PKC isoforms are determined by the DAG affinity and conformational flexibility of their C1 domains.",
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