title: The interaction of protein kinase C isozymes alpha, iota, and theta with the cytoplasmic domain of L-selectin is modulated by phosphorylation of the receptor.
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 Ca(2+)-independent PKC isozymes theta and iota 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 PKCalpha and -theta 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 PKCalpha and -theta 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.
authors.affiliation: Institut für Klinische Chemie und Pathobiochemie, Charité, Berlin 12200, Germany. karin.kilian@charite.de
authors.forename: Karin
authors.initials: K
authors.lastname: Kilian
authors.forename: Jens
authors.initials: J
authors.lastname: Dernedde
authors.forename: Eva-Christina
authors.initials: EC
authors.lastname: Mueller
authors.forename: Inke
authors.initials: I
authors.lastname: Bahr
authors.forename: Rudolf
authors.initials: R
authors.lastname: Tauber
mesh_terms: D000595:Amino Acid Sequence; D017252:CD3 Complex / Q000096:biosynthesis; D002118:Calcium / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D006152:Cyclic GMP / Q000378:metabolism; D003593:Cytoplasm / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005786:Gene Expression Regulation; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D007249:Inflammation; D007527:Isoenzymes / Q000378:metabolism*; D019169:Jurkat Cells; D019041:L-Selectin / Q000737:chemistry* / Q000378:metabolism*; D008214:Lymphocytes / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D000074121:Protein Kinase C-theta; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D015398:Signal Transduction; D013912:Threonine / Q000737:chemistry; D000099293:Protein Kinase C-lambda
chemical_list: D017252:CD3 Complex; D002118:Calcium; D003432:Cross-Linking Reagents; D006152:Cyclic GMP; D005982:Glutathione Transferase; D007527:Isoenzymes; D019041:L-Selectin; D020033:Protein Isoforms; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D000074121:Protein Kinase C-theta; D011993:Recombinant Fusion Proteins; D012694:Serine; D013912:Threonine; D000099293:Protein Kinase C-lambda; C496628:PRKCA protein, human; C076233:PRKCQ protein, human