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The interaction of protein kinase C isozymes α, ι, and θ with the cytoplasmic domain of L-selectin is modulated by phosphorylation of the receptor

Item Type:Article
Title:The interaction of protein kinase C isozymes α, ι, and θ with the cytoplasmic domain of L-selectin is modulated by phosphorylation of the receptor
Creators Name:Kilian, K. and Dernedde, J. and Mueller, E.C. and Bahr, I. and Tauber, R.
Abstract:The leukocyte adhesion molecule L-selectin has an important role in the initial steps of leukocyte extravasation during inflammation and lymphocyte homing. Its cytoplasmic domain is involved in signal transduction after L-selectin cross-linking and in the regulation of receptor binding activity in response to intracellular signals. However, the signaling events occurring at the level of the receptor are largely unknown. This study therefore addressed the question of whether protein kinases associate with the cytoplasmic domain of the receptor and mediate its phosphorylation. Using a glutathione S-transferase fusion protein of the L-selectin cytoplasmic domain, we isolated a kinase activity from cellular extracts of the human leukemic Jurkat T-cell line that phosphorylated L-selectin on serine residues. This kinase showed characteristics of the protein kinase C (PKC) family. Moreover, the Ca2+-independent PKC isozymes θ and ι were found associated with the cytoplasmic domain of L-selectin. Pseudosubstrate inhibitors of these isozymes abolished phosphorylation of the cytoplasmic domain, demonstrating that these kinases are responsible for the phosphorylation. Analysis of proteins specifically bound to the phosphorylated cytoplasmic tail of L-selectin revealed that PKCα and -θ are strongly associated with the phosphorylated cytoplasmic domain of L-selectin. Binding of these isozymes to L-selectin was also found in intact cells after phorbol ester treatment inducing serine phosphorylation of the receptor. Furthermore, stimulation of Jurkat T-cells by CD3 cross-linking induced association of PKCα and -θ with L-selectin, indicating a role of these kinases in the regulation of L-selectin through the T-cell receptor complex. The phosphorylation-regulated association of PKC isozymes with the cytoplasmic domain of L-selectin indicates an important role of this kinase family in L-selectin signal transduction.
Keywords:Amino Acid Sequence, Amino Acid Sequence Homology, Calcium, CD3 Antigens, Cross-Linking Reagents, Cyclic GMP, Cytoplasm, Drug Dose-Response Relationship, Gene Expression Regulation, Glutathione Transferase, Inflammation, Isoenzymes, Jurkat Cells, L-Selectin, Lymphocytes, Mass Spectrometry, Molecular Sequence Data, Phosphorylation, Polyacrylamide Gel Electrophoresis, Precipitin Tests, Protein Binding, Protein Isoforms, Protein Kinase C, Protein Kinase C-Alpha, Recombinant Fusion Proteins, Serine, Signal Transduction, Tertiary Protein Structure, Threonine
Source:Journal of Biological Chemistry
Publisher:American Society for Biochemistry and Molecular Biology
Page Range:34472-34480
Date:1 January 2004
Official Publication:https://doi.org/10.1074/jbc.M405916200
PubMed:View item in PubMed

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