The carboxyl-terminal domain of Atypical protein kinase Cζ binds to ceramide and regulates junction formation in Epithelial cells

Guanghu Wang, Kannan Krishnamurthy, Nagavedi S. Umapathy, Alexander D. Verin, Erhard Bieberich

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Atypical protein kinase Cs (PKCs) (aPKCζ and λ/ι) have emerged as important binding partners for ceramide, a membrane-resident cell signaling lipid that is involved in the regulation of apoptosis as well as cell polarity. Using ceramide overlay assays with proteolytic fragments of PKCζ and vesicle binding assays with ectopically expressed protein, we show that a protein fragment comprising the carboxyl-terminal 20-kDa sequence of PKCζ (C20ζ, amino acids 405-592) bound to C16:0 ceramide. This sequence is not identical to the C1 domain (amino acids 131-180), which has been suggested to serve as a potential ceramide binding domain. Using immunocytochemistry, we found that a C20ζ protein fragment ectopically expressed in two epithelial cell types (neural progenitors and Madin-Darby canine kidney cells) co-distributed with ceramide. Stable expression of C20ζ-EGFP in Madin-Darby canine kidney cells disrupted the formation of adherens and tight junctions and impaired the epithelium integrity by reducing transepithelial electrical resistance. Disruption of cell adhesion and loss of transepithelial electrical resistance was prevented by incubation with C16:0 ceramide. Our results show, for the first time, that there is a novel ceramide binding domain (C20ζ) in the carboxyl terminus of aPKC. Our results also show that the interaction of ceramide with this binding domain is essential for cell-to-cell contacts in epithelia. Therefore, ceramide interaction with the C20ζ binding domain is a potential mechanism by which ceramide and aPKC regulate the formation of junctional complexes in epithelial cells.

Original languageEnglish (US)
Pages (from-to)14469-14475
Number of pages7
JournalJournal of Biological Chemistry
Volume284
Issue number21
DOIs
StatePublished - May 22 2009

Fingerprint

Ceramides
Epithelial Cells
Protein Kinases
Madin Darby Canine Kidney Cells
Electric Impedance
Acoustic impedance
Epithelium
Assays
Adherens Junctions
Amino Acids
Cell Polarity
Proteins
PKC-3 protein
Tight Junctions
Cell signaling
Cell Adhesion
Cell adhesion
Immunohistochemistry
Cell Membrane
Apoptosis

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology
  • Molecular Biology

Cite this

The carboxyl-terminal domain of Atypical protein kinase Cζ binds to ceramide and regulates junction formation in Epithelial cells. / Wang, Guanghu; Krishnamurthy, Kannan; Umapathy, Nagavedi S.; Verin, Alexander D.; Bieberich, Erhard.

In: Journal of Biological Chemistry, Vol. 284, No. 21, 22.05.2009, p. 14469-14475.

Research output: Contribution to journalArticle

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