p56Lck anchors CD4 to distinct microdomains on microvilli

Abstract

Cell-surface microvilli play a central role in adhesion, fusion, and signaling processes. Some adhesion and signaling receptors segregate on microvilli but the determinants of this localization remain mostly unknown. In this study, we considered CD4, a receptor involved in immune response and HIV infection, and p56(Lck), a CD4-associated tyrosine kinase. Analysis of CD4 trafficking reveals that p56(Lck) binds tightly to CD4 independently of its activation state and inhibits CD4 internalization. Electron microscopy analysis established that p56(Lck) mediates CD4 association with microvilli whereas biochemical data indicate that p56(Lck) expression renders CD4 insoluble by the nonionic detergent Triton X-100. In addition, cytoskeleton-disrupting agent increased CD4 solubility, suggesting the involvement of cytoskeletal elements in CD4 anchoring to microvilli. This concept was supported further by the observation that the lateral mobility of CD4 within the plasma membrane was decreased in cells expressing p56(Lck). Finally, isolation of detergent-resistant membranes revealed that the complex CD4-p56(Lck) is enriched within these domains as opposed to conditions in which CD4 does not interact with p56(Lck). In conclusion, our results show that p56(Lck) targets CD4 to specialized lipid microdomains preferentially localized on microvilli. This localization, which prevents CD4 internalization, might facilitate CD4-mediated adhesion processes and could correspond to the signaling site of the receptor.

Figure 1

p56^Lck prevents CD4 endocytosis independently of the kinase activation state. (A) Rates of CD4 internalization were calculated based on the percentage of the total cell-associated radioactivity incorporated after 5 min at 37°C by using iodinated anti-CD4 antibodies as described in Materials and Methods. (B) Kinase activity of p56^Lck mutants was assessed in a kinase assay monitoring phosphorylation of enolase as a substrate. As a control of the reaction, 2 μM PP2 was used to inhibit the reaction. Data are representative of three independent experiments. (C) Internalization of CD4 in 293T cells expressing p56^Lck mutants by using a FACS-based assay as described in Materials and Methods. Data are means ± SE of four independent experiments. (D) p56^Lck association with CD4 in 293T cells cotransfected with CD4 and p56^Lck mutants. Data are representative of three independent experiments.

Figure 2

p56^Lck binding to CD4 triggers CD4 anchoring to microvilli. (A) Electron micrograph showing CD4 radiolabeling on microvilli in CEM cells. (Bar = 0.2 μm.) (B) Kinetics of radiolabeled CD4 association with microvilli in CEM and HL60 cells. Data are means ± SE from two to three separate experiments totaling 900–1,200 autoradiographic grains for each time point. (C) p56^Lck association with CD4 in CEM cells treated with 1,10-O-phenanthroline for 30 min at 37°C before cell lysis. Data are representative of three independent experiments. (D) Electron micrograph showing CD4 gold labeling on microvilli in CEM cells. (Bar = 0.2 μm.) (E) Quantitation of gold-labeled CD4 association with microvilli at 4°C in CEM cells treated or not treated with 10 mM 1,10-O-phenanthroline and HL60 cells. Data are means ± SE from three to four separate experiments totaling 78 cells per 2,483 gold particles, 40 cells per 1,119 gold particles, and 81 cells per 1,929 gold particles counted for CEM cells, CEM cells treated with 1,10-O-phenanthroline, and HL60 cells, respectively.

Figure 3

Disruption of the cytoskeleton increases CD4 TX100 solubility in p56^Lck-expressing cells. (A) Western analysis of CD4 and p56^Lck solubility by TX100. Cells were lysed in buffer containing 1% TX100 for 20 min at 4°C and then centrifuged at 100,000 × g for 1 h at 4°C to fractionate the lysates in a TX100-soluble (S) and TX100-insoluble (P) fraction. Data are representative of three independent experiments. (B) Typical FACS profile of CD4-associated fluorescence in CEM cells ± 1% TX100 and cytochalasin D (Cyto. D). (C) Quantitation of CD4 solubility by TX100 in CEM and HL60 cells treated or not treated with Cyto. D. Data are expressed as the ratio of fluorescence associated with cells after TX100 addition to fluorescence associated with cells before TX100 addition. Results are means ± SE from three to four independent experiments.

Figure 4

Lateral mobility of CD4 is restricted in p56^Lck-expressing cells. The lateral diffusion of CD4 was measured at 37°C in individual, cultured CEM cells and HL60 cells by using FRAP analysis. (A) Mean diffusion constant. (B) Mobile fraction. Data are means ± SE of 35–59 cells analyzed for each condition.

Figure 5

p56^Lck triggers CD4 segregation in DRMs. DRMs were prepared from CEM and HL60 cells as described in Materials and Methods and fractions were analyzed by SDS/PAGE and Western blotting. (A) Typical distribution of CD4, p56^Lck, CD71, and AlkP is shown. Data are representative of three independent experiments. (B) Disruption of the CD4-p56^Lck complex by 1,10-O-phenanthroline redistributes CD4, but not p56^Lck, out of DRMs. CEM cells were treated for 30 min at 37°C with 1,10-O-phenanthroline before starting the fractionation procedure, and distribution of CD4 and p56^Lck was analyzed. Data are representative of three independent experiments. (C) CD4 is palmitoylated in both CEM and HL60 cells. Cells were metabolically labeled with [³H]palmitate, and CD4 palmitoylation was revealed as described in Materials and Methods. Data are representative of three independent experiments.