ABSTRACT
The SH4 domain of Src family of nonreceptor protein tyrosine kinases represents the extreme N-terminal 1-16 amino acid region which mediates membrane association of these proteins and facilitates their functions. The SH4 domains among Src members lack well-defined sequence consensus and vary in the net charge. However, they readily anchor to the cytoplasmic face of the plasma membrane upon fatty acid acylation. Here, we report the membrane association of differentially acylated SH4 domain of Lck kinase, which has net negative charge at physiological pH. Our results suggest that despite the net negative charge, the SH4 domain of Lck associates with membranes upon fatty acid acylation. While myristoylation at the N-terminus is sufficient for providing membrane anchorage, multiple acylation determines orientation of the peptide chain with respect to the lipid bilayer. Hence, fatty acylation serves more than just a lipid anchor. It has an important role in regulating the spatial orientation of the peptide domain with respect to the lipid bilayer, which could be important for the interaction of the other domains of these kinases with their partners.
Subject(s)
Fatty Acids/chemistry , Lymphocyte Specific Protein Tyrosine Kinase p56(lck)/chemistry , Membrane Lipids/chemistry , Peptides/chemistry , Unilamellar Liposomes/chemistry , Acylation , Amino Acid Sequence , Binding Sites , Fluorescence Resonance Energy Transfer , Molecular Sequence Data , Myristic Acid/chemistry , Peptides/chemical synthesis , Protein Binding , Protein Structure, Tertiary , Solid-Phase Synthesis Techniques , Static ElectricityABSTRACT
Lipopeptides derived from protein kinase C (PKC) pseudosubstrates have the ability to cross the plasma membrane in cells and modulate the activity of PKC in the cytoplasm. Myristoylation or palmitoylation appears to promote translocation across membranes, as the non-acylated peptides are membrane impermeant. We have investigated, by fluorescence spectroscopy, how myristoylation modulates the interaction of the PKC pseudosubstrate peptide KSIYRRGARRWRKL with lipid vesicles and translocation across the lipid bilayer. Our results indicate that myristoylated peptides are intimately associated with lipid vesicles and are not peripherally bound. When visualized under a microscope, myristoylation does appear to facilitate translocation across the lipid bilayer in multilamellar lipid vesicles. Translocation does not involve large-scale destabilization of the bilayer structure. Myristoylation promotes translocation into the hydrophobic interior of the lipid bilayer even when the non-acylated peptide has only weak affinity for membranes and is also only peripherally associated with lipid vesicles.
Subject(s)
Lipid Bilayers/chemistry , Lipids/chemistry , Myristic Acid/chemistry , Protein Kinase C/chemistry , Cell Membrane/metabolism , Chromatography, Gel , Cytoplasm/metabolism , Diffusion , Dose-Response Relationship, Drug , Fluorescence Resonance Energy Transfer , Iodides/chemistry , Microscopy, Fluorescence , Myristic Acids/chemistry , Peptides/chemistry , Protein Binding , Protein Structure, Tertiary , Protein Transport , Spectrometry, Fluorescence , Time Factors , Tryptophan/chemistryABSTRACT
Covalent modification with fatty acids is observed in several proteins that play crucial roles in cellular physiology. In this paper, a convenient method for the generation of multiple fatty acylated synthetic peptides is described. Peptides were synthesized using solid phase procedures with fluorenylmethoxycarbonyl a-amino protected amino acids. Acetamidomethyl protected cysteines were employed. The thiol protecting group was selectively deprotected and acylation was carried out on the resin-bound peptides. The strategy described in this report is applicable to any peptide sequence.
