Determinants of membrane association in the SH4 domain of Fyn: roles of N-terminus myristoylation and side-chain thioacylation.
The SH4 domain of Fyn, a member of the Src family of tyrosine kinases, though rich in polar amino acid residues, anchors to the cytosolic face of membranes upon fatty acylation. In order to probe the requirement of specific fatty acylation at the N-terminus and at the side-chain of this domain for membrane-association, we have studied the interaction of peptides corresponding to the polar segment of the SH4 domain of Fyn and its mono- and dually fatty acylated analogs with model membranes. While the polar segment without covalently linked fatty acids (KDKEATKLTEW-amide) does not interact with lipid vesicles, peptides with one covalently linked fatty acid at the N-terminus or in the side-chain, associate with zwitterionic and anionic lipids to varying degrees. The interaction of dually acylated peptides (Myr-GK(epsilon-myr)KDKEATKLTEW-amide and Myr-GC(S-pal)KDKEATKLTEW-amide) with lipids depends on the linkage between fatty acyl side-chain and peptide backbone. The peptide chain associates with membranes only when the side-chain acylation is via an amide bond and not via a thioester bond. Our investigations indicate that acylation is essential for membrane targeting and unacylated polar stretch of the SH4 domain does not have a role in membrane-anchoring. Side-chain acylation via a thioester bond not only provides membrane anchorage but also directs the peptide chain away from the bilayer which might be important to enable the full length protein to interact with other signaling partners.
Rawat
Anoop
A
Centre for Cellular and Molecular Biology, Council of Scientific and Industrial Research, Uppal Road, Hyderabad 500 007, India.
Nagaraj
Ramakrishnan
R
D000595:Amino Acid Sequence; D001665:Binding Sites; D005227:Fatty Acids / Q000737:chemistry / Q000378:metabolism; D008051:Lipid Bilayers / Q000737:chemistry* / Q000378:metabolism; D008563:Membrane Lipids / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D019814:Myristic Acid / Q000737:chemistry / Q000378:metabolism; D010455:Peptides / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D011485:Protein Binding; D051076:Proto-Oncogene Proteins c-fyn / Q000737:chemistry*; D013050:Spectrometry, Fluorescence; D013438:Sulfhydryl Compounds / Q000737:chemistry / Q000378:metabolism; D013499:Surface Properties
D005227:Fatty Acids; D008051:Lipid Bilayers; D008563:Membrane Lipids; D010455:Peptides; D013438:Sulfhydryl Compounds; D019814:Myristic Acid; D051076:Proto-Oncogene Proteins c-fyn