Diacylglycerol-induced membrane targeting and activation of protein kinase Cε

Mechanistic differences between protein kinases Cδ and Cε

Robert Stahelin, Michelle A. Digman, Martina Medkova, Bharath Ananthanarayanan, Heather R. Melowic, John D. Rafter, Wonhwa Cho

Research output: Contribution to journalArticle

86 Citations (Scopus)

Abstract

Two novel protein kinases C (PKC), PKCδ and PKCε, have been reported to have opposing functions in some mammalian cells. To understand the basis of their distinct cellular functions and regulation, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKCε and compared it with, that of PKCδ. The regulatory domains of novel PKC contain a C2 domain and a tandem repeat of C1 domains (C1A and C1B), which have been identified as the interaction site for DAG and phorbol ester. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCε have comparably high affinities for DAG and phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCε mutants showed that both the C1A and C1B domains play a role in the DAG-induced membrane binding and activation of PKCε. The C1 domains of PKCε are not conformationally restricted and readily accessible for DAG binding unlike those of PKCε. Consequently, phosphatidylserine-dependent unleashing of C1 domains seen with PKCδ was not necessary for PKCε. Cell studies with fluorescent protein-tagged PKCδ showed that, due to the lack of lipid headgroup selectivity, PKCε translocated to both the plasma membrane and the nuclear membrane, whereas PKCδ migrates specifically to the plasma membrane under the conditions in which DAG is evenly distributed among intracellular membranes of HEK293 cells. Also, PKCε translocated much faster than PKCε due to conformational flexibility of its C1 domains. Collectively, these results provide new insight into the differential activation mechanisms of PKCδ and PKCε based on different structural and functional properties of their C1 domains.

Original languageEnglish (US)
Pages (from-to)19784-19793
Number of pages10
JournalJournal of Biological Chemistry
Volume280
Issue number20
DOIs
StatePublished - May 20 2005
Externally publishedYes

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Diglycerides
Protein Kinase C
Chemical activation
Membranes
Membrane Proteins
Carrier Proteins
Phorbol Esters
Cell membranes
Cell Membrane
Intracellular Membranes
Calorimetry
Tandem Repeat Sequences
Surface Plasmon Resonance
HEK293 Cells
Phosphatidylserines
Nuclear Envelope
Surface plasmon resonance

ASJC Scopus subject areas

  • Biochemistry

Cite this

Diacylglycerol-induced membrane targeting and activation of protein kinase Cε : Mechanistic differences between protein kinases Cδ and Cε. / Stahelin, Robert; Digman, Michelle A.; Medkova, Martina; Ananthanarayanan, Bharath; Melowic, Heather R.; Rafter, John D.; Cho, Wonhwa.

In: Journal of Biological Chemistry, Vol. 280, No. 20, 20.05.2005, p. 19784-19793.

Research output: Contribution to journalArticle

Stahelin, Robert ; Digman, Michelle A. ; Medkova, Martina ; Ananthanarayanan, Bharath ; Melowic, Heather R. ; Rafter, John D. ; Cho, Wonhwa. / Diacylglycerol-induced membrane targeting and activation of protein kinase Cε : Mechanistic differences between protein kinases Cδ and Cε. In: Journal of Biological Chemistry. 2005 ; Vol. 280, No. 20. pp. 19784-19793.
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