The dyslexia-associated protein KIAA0319 interacts with adaptor protein 2 and follows the classical clathrin-mediated endocytosis pathway

Abstract

Recently, genetic studies have implicated KIAA0319 in developmental dyslexia, the most common of the childhood learning disorders. The first functional data indicated that the KIAA0319 protein is expressed on the plasma membrane and may be involved in neuronal migration. Further analysis of the subcellular distribution of the overexpressed protein in mammalian cells indicates that KIAA0319 can colocalize with the early endosomal marker early endosome antigen 1 (EEA1) in large intracellular vesicles, suggesting that it is endocytosed. Antibody internalization assays with full-length KIAA0319 and deletion constructs confirmed that KIAA0319 is internalized and showed the importance of the cytoplasmic juxtamembranal region in this process. The present study has identified the medium subunit (mu2) of adaptor protein 2 (AP-2) as a binding partner of KIAA0319 in a yeast two-hybrid screen. Using Rab5 mutants or depletion of the mu-subunit of AP-2 or clathrin heavy chain by RNA interference, we demonstrate that KIAA0319 follows a clathrin-mediated endocytic pathway. We also identify tyrosine-995 of KIAA0319 as a critical amino acid required for the interaction with AP-2 and subsequent internalization. These results suggest the surface expression of KIAA0319 is regulated by endocytosis, supporting the idea that the internalization and recycling of the protein may be involved in fine tuning its role in neuronal migration.

Fig. 1.

KIAA0319 is internalized into early endosomes. Transiently transfected HEK293T cells expressing mycHIS-tagged full-length KIAA0319 KA (A–L) or several COOH-terminal-deletion proteins (M–O) were processed for normal IF or for internalization assays as described in materials and methods. KIAA0319 proteins were detected with polyclonal R2 antiserum and/or monoclonal anti-myc 9E10 antibody. KA is detected in vesicles (A–C), some of which show colocalization with the endosomal marker anti-EEA1 (D–F) (arrows). For the internalization assays, cells were loaded at 4°C with R2 antiserum, followed by incubation at 37°C to allow endocytosis to proceed (G–L). Antibody bound at 4°C stays at the plasma membrane (G–I) but is internalized after incubation at 37°C (J–L). Similar internalization assays performed for deletion proteins Kd20-21 (M), Kd20-21a (N), and Kd21b (O) (lacking residues 984-1072, 984-1023, and 1031-1072, respectively) show that only Kd21b is internalized. This indicates that the juxtamembranal region of the cytoplasmic domain contains a signal for endocytosis (see supplementary Fig. 1 for more details). 10-μm bars are shown in the merged panels.

Fig. 2.

Effect of Rab5 mutants on KIAA0319 internalization. HEK293T cells coexpressing mycHIS-tagged full-length KIAA0319 and either the constitutively active Rab5 Q79L mutant (A–F) or the dominant negative Rab5 S34N mutant (G–L) were subjected to R2 antiserum internalization assays. Rab5 mutants are detected by their GFP tag (A, D, G, J). As shown in Fig. 1, R2 antiserum bound at 4°C stays at the plasma membrane (B, H). In Rab5 Q79L transfected cells (D), KIAA0319 is internalized (E) and colocalizes with the enlarged Rab5-positive endosomes (F) after incubation at 37°C. In cells overexpressing Rab5 S34N (J), the R2 antiserum stays at the plasma membrane after the incubation at 37°C (K), and no colocalization with the Rab5 mutant is detected (L), suggesting that the endocytosis of KIAA0319 is inhibited. 10-μm bars are shown in the merge panels.

Fig. 3.

KIAA0319 interacts with the μ-subunit of adaptor-2 (AP-2). A: yeast two-hybrid (Y2H) analysis. Results of initial library screening by mating (top) and reconfirmation assay (bottom). Yeast Y190 was cotransformed with the prey plasmid encoding μ2 (pGADT7_μ2) and the bait plasmid (pAS2_KACt) or a negative control plasmid (pLAM5′-1) and grown on selective media lacking tryptophan, leucine, and histidine and supplemented with 20 mM 3-amino-1,2,4-triazole (3-AT). Only yeast containing plasmids encoding KACt and μ2 could activate the reporter gene his and grow on selective media (assays shown in duplicate). B: sequence of the cytoplasmic domain of KIAA0319 (KACt) indicating exons, fragments f1, f2, and f3, and position of tyrosine 995 (see text and Fig. 4). C: GST-pulldown assays. HeLa cell protein lysates were incubated with GST or GST-fusion proteins (full-length KACt or overlapping fragments f1-f2-f3) immobilized on glutathione sepharose beads. Eluates were analyzed by Western blot using anti-AP50 antibody, against the μ2-subunit of AP-2. A Ponceau staining of the membrane indicates the expression level of GST and GST-fusion proteins used in the experiment. Only KACt and f1 GST-fusion proteins are able to pull down the μ2 subunit of AP-2.

Fig. 4.

KIAA0319 undergoes clathrin-mediated endocytosis. HeLa cells were treated with transfection reagent alone (mock), with 100 nM scrambled small interfering RNA (siRNA) or with 100 nM siRNA directed against either μ2 or cathrin heavy chain (CLTC). After siRNA treatment, cells were transfected with a full-length KIAA0319 construct containing a V5 tag. A: cell lysates were prepared 72 h later and Western blotted using antibodies directed against μ2, CLTC, or actin. RNAi is specific for μ2 and CTLC and does not influence actin levels. B: R2 internalization assays in siRNA-treated cells. KIAA0319 protein at the plasma membrane was labeled with anti-KIAA0319 R2 antiserum, and after a period of internalization cells were fixed and processed to detect internalized R2 (B, E, H, K). Cells silenced for μ2 or CLTC can be identified by the absence of transferrin-594 uptake (A, D, G, J). In mock transfected and scrambled control cells, R2-labeled KIAA0319 is observed intracellularly, where it colocalizes with transferrin (A–F). In cells depleted of μ2 or CLTC, R2-labeled KIAA0319 is observed only on the cell surface (H, K). The arrowheads indicate some nondepleted cells that can act as an internal negative control. 10-μm bars are shown in the merged panels.

Fig. 5.

Tyrosine-995 is needed for the endocytosis of KIAA0319. A: effect of Y995A mutation on the subcellular localization of KIAA0319. HeLa cells were transfected with constructs expressing either wild-type (KAv) (A–C) or mutant (KAv-Y995A) (D–F) V5-tagged full-length KIAA0319 protein. Proteins were detected with anti-V5 antibody (A, D) or by R2 antiserum internalization assays (B, E). KAv is observed on the cell surface and in internal structures, whereas KAv-Y995A is found only at the cell surface and R2 antibodies are not internalized. Internalization of transferrin-594 is unaffected by the expression of either construct demonstrating clathrin-dependent endocytosis occurs normally in both cases (C, F). b: effect of Y995A mutation on the interaction between KIAA0319 COOH-terminus and the μ2-subunit by Y2H analysis. Y190 yeast cells were cotransformed with either the bait vectors pAS2_KACt (wild-type bait) or pAS2_KACt-Y995A (mutated bait) and the prey plasmid encoding μ2 (pGADT7_μ2). Only yeast transformed with wild-type KACt could activate the reporter gene his and grow on media lacking histidine (assays shown in duplicate). c: effect of Y995A mutation on the interaction between KIAA0319 and the μ2-subunit by GST-pulldown analysis. Experiments performed as described in Fig. 3. Binding of μ2 was highly reduced with mutated KACt fragment (KACt-Y995A) compared with wild-type fragment. Ponceau staining indicates amounts of fusion protein used for the GST pulldown. Scale bars: 10 μm.