Signals determining protein tyrosine kinase and glycosyl-phosphatidylinositol-anchored protein targeting to a glycolipid-enriched membrane fraction.
Glycosyl-phosphatidylinositol (GPI)-anchored membrane proteins and certain protein tyrosine kinases associate with a Triton X-100-insoluble, glycolipid-enriched membrane fraction in MDCK cells. Also, certain protein tyrosine kinases have been shown to associate with GPI-anchored proteins in other cell types. To characterize the interaction between GPI-anchored proteins and protein tyrosine kinases, GPI-anchored proteins were coexpressed with p56lck in HeLa cells. Both proteins were shown to target independently to the glycolipid-enriched membranes. Coimmunoprecipitation of GPI-anchored proteins and p56lck occurred only when both proteins were located in the glycolipid-enriched membranes, and gentle disruption of these membranes abolished the interaction. The GPI anchor was found to be the targeting signal for this membrane fraction in GPI-anchored proteins. Analysis of mutants indicated that p56lck was nearly quantitatively palmitoylated at Cys-5 but not palmitoylated at Cys-3. The nonpalmitoylated cysteine at position 3 was very important for association of p56lck with the membrane fraction, while palmitoylation at Cys-5 promoted only a low level of interaction. Because other src family protein tyrosine kinases that are associated with GPI-anchored proteins always contain a Cys-3, we propose that this residue, in addition to the N-terminal myristate, is part of a common signal targeting these proteins to a membrane domain that has been linked to transmembrane signaling.
Department of Pathology, Yale University School of Medicine, New Haven, Connecticut.
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Rodgers
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Crise
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Rose
D000595:Amino Acid Sequence; D001483:Base Sequence; D002451:Cell Compartmentation; D017931:DNA Primers / Q000737:chemistry; D006017:Glycolipids / Q000378:metabolism*; D017261:Glycosylphosphatidylinositols / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck); D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009226:Myristates / Q000378:metabolism; D010168:Palmitates / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship
D017931:DNA Primers; D006017:Glycolipids; D017261:Glycosylphosphatidylinositols; D009226:Myristates; D010168:Palmitates; D011506:Proteins; C055324:phospholipase A2-activating protein; D011505:Protein-Tyrosine Kinases; D019860:Lymphocyte Specific Protein Tyrosine Kinase p56(lck)