Differently anchored influenza hemagglutinin mutants display distinct interaction dynamics with mutual rafts

Lipid rafts play important roles in cellular functions through concentrating or sequestering membrane proteins. This requires proteins to differ in the stability of their interactions with lipid rafts. However, knowledge of the dynamics of membrane protein–raft interactions is lacking. We employed FRAP to measure in live cells the lateral diffusion of influenza hemagglutinin (HA) proteins that differ in raft association. This approach can detect weak interactions with rafts not detectable by biochemical methods. Wild-type (wt) HA and glycosylphosphatidylinositol (GPI)-anchored HA (BHA-PI) diffused slower than a nonraft HA mutant, but became equal to the latter after cholesterol depletion. When antigenically distinct BHA-PI and wt HA were coexpressed, aggregation of BHA-PI into immobile patches reduced wt HA diffusion rate, suggesting transient interactions with BHA-PI raft patches. Conversely, patching wt HA reduced the mobile fraction of BHA-PI, indicating stable interactions with wt HA patches. Thus, the anchoring mode determines protein–raft interaction dynamics. GPI-anchored and transmembrane proteins can share the same rafts, and different proteins can interact stably or transiently with the same raft domains

Src kinase activity and SH2 domain regulate the dynamics of Src association with lipid and protein targets

Src functions depend on its association with the plasma membrane and with specific membrane-associated assemblies. Many aspects of these interactions are unclear. We investigated the functions of kinase, SH2, and SH3 domains in Src membrane interactions. We used FRAP beam-size analysis in live cells expressing a series of c-Src–GFP proteins with targeted mutations in specific domains together with biochemical experiments to determine whether the mutants can generate and bind to phosphotyrosyl proteins. Wild-type Src displays lipid-like membrane association, whereas constitutively active Src-Y527F interacts transiently with slower-diffusing membrane-associated proteins. These interactions require Src kinase activity and SH2 binding, but not SH3 binding. Furthermore, overexpression of paxillin, an Src substrate with a high cytoplasmic population, competes with membrane phosphotyrosyl protein targets for binding to activated Src. Our observations indicate that the interactions of Src with lipid and protein targets are dynamic and that the kinase and SH2 domain cooperate in the membrane targeting of Src

Clustering of Raft-Associated Proteins in the External Membrane Leaflet Modulates Internal Leaflet H-Ras Diffusion and Signaling

One of the least-explored aspects of cholesterol-enriched domains (rafts) in cells is the coupling between such domains in the external and internal monolayers and its potential to modulate transbilayer signal transduction. Here, we employed fluorescence recovery after photobleaching to study the effects of antibody-mediated patching of influenza hemagglutinin (HA) proteins [raft-resident wild-type HA and glycosylphosphatidylinositol-anchored HA, or the nonraft mutant HA(2A520)] on the lateral diffusion of internal-leaflet raft and nonraft Ras isoforms (H-Ras and K-Ras, respectively). Our studies demonstrate that the clustering of outer-leaflet or transmembrane raft-associated HA proteins (but not their nonraft mutants) retards the lateral diffusion of H-Ras (but not K-Ras), suggesting stabilized interactions of H-Ras with the clusters of raft-associated HA proteins. These modulations were paralleled by specific effects on the activity of H-Ras but not of the nonraft K-Ras. Thus, clustering raft-associated HA proteins facilitated the early step whereby H-Ras is converted to an activated, GTP-loaded state but inhibited the ensuing step of downstream signaling via the Mek/Erk pathway. We propose a model for the modulation of transbilayer signaling by clustering of raft proteins, where external clustering (antibody or ligand mediated) enhances the association of internal-leaflet proteins with the stabilized clusters, promoting either enhancement or inhibition of signaling