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Differential requirements for AP-2 in clathrin-mediated endocytosis.

Conner SD, Schmid SL - J. Cell Biol. (2003)

Bottom Line: Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit.Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo.Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2.

View Article: PubMed Central - PubMed

Affiliation: The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought.

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AP-2–deficient clathrin-coated pits are functional for EGF uptake. (A) WT AAK1-infected tTA HeLa cells (cultured as described in the legend to Fig. 3 C) show no defect in the recruitment of clathrin to the plasma membrane or its ability to generate coated pits, as observed by immunolocalization using the mAb X22. (B) EGF internalization was tested in tTA HeLa cells infected with adenoviruses expressing either tTA, WT, or K74A AAK1, or K44A dynamin-1 adenoviruses, as indicated in the legend. Protein expression levels were tested by immunoblot analysis (inset) using pAbs against either the ΔAID AAK1 fragment (lanes 1–3) or against dynamin (lanes 1 and 4). (C) tTA HeLa cells, cultured on coverslips and infected with the indicated adenoviruses, were tested for their ability to internalize rhodamine-conjugated Tfn and Alexa-488–conjugated EGF, simultaneously (Molecular Probes). Cells were incubated in the presence of 2 ng/ml EGF and 4 μg/ml Tfn for 15 min at 37°C. Cells were then transferred to ice, washed to remove unbound ligand, fixed with acetone, and visualized by epifluorescence as described in the legend to Fig. 3. A significant decrease in Tfn accumulation in the endosome is observed in WT AAK1–infected cells relative to AID AAK1– or control-infected cells (arrows), whereas EGF uptake is unaffected.
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fig4: AP-2–deficient clathrin-coated pits are functional for EGF uptake. (A) WT AAK1-infected tTA HeLa cells (cultured as described in the legend to Fig. 3 C) show no defect in the recruitment of clathrin to the plasma membrane or its ability to generate coated pits, as observed by immunolocalization using the mAb X22. (B) EGF internalization was tested in tTA HeLa cells infected with adenoviruses expressing either tTA, WT, or K74A AAK1, or K44A dynamin-1 adenoviruses, as indicated in the legend. Protein expression levels were tested by immunoblot analysis (inset) using pAbs against either the ΔAID AAK1 fragment (lanes 1–3) or against dynamin (lanes 1 and 4). (C) tTA HeLa cells, cultured on coverslips and infected with the indicated adenoviruses, were tested for their ability to internalize rhodamine-conjugated Tfn and Alexa-488–conjugated EGF, simultaneously (Molecular Probes). Cells were incubated in the presence of 2 ng/ml EGF and 4 μg/ml Tfn for 15 min at 37°C. Cells were then transferred to ice, washed to remove unbound ligand, fixed with acetone, and visualized by epifluorescence as described in the legend to Fig. 3. A significant decrease in Tfn accumulation in the endosome is observed in WT AAK1–infected cells relative to AID AAK1– or control-infected cells (arrows), whereas EGF uptake is unaffected.

Mentions: The μ2 subunit of AP-2 specifically recognizes tyrosine-based internalization motifs, whereas other AP-2 subunits are believed to function in endocytosis by directing clathrin assembly into curved lattices and by recruiting other essential cofactors to the coated pit (Kirchhausen, 1999). Therefore, we expected that clathrin-coated pit assembly would also be disrupted in cells overexpressing inhibitory AAK1 constructs that functionally sequester AP-2. Surprisingly, clathrin recruitment into coated pits was not altered in WT AAK1–overexpressing cells relative to controls (Fig. 4 A). Previous studies have established that EGF and TfnRs are internalized in the same coated pits (Lamaze et al., 1993). We therefore asked if AAK1 overexpression had any effect on EGF uptake. Surprisingly, neither WT nor K74A AAK1 overexpression had any effect on the internalization of EGF compared with controls (Fig. 4 , B and C). High concentrations of EGF are known to saturate the clathrin-mediated pathway for EGFR endocytosis (Jiang and Sorkin, 2003); therefore, care was taken to use low concentrations (2 ng/ml) of 125I-labeled EGF for these assays. As an additional control for CME, cells infected with recombinant K44A dynamin-1 adenovirus showed the expected EGF internalization defect (Damke et al., 1994). We cannot rule out that the small amounts of AP-2 remaining at the cell surface are selectively associated with coated pits engaged in EGF uptake. However, our results are completely consistent with recent findings reporting that siRNA-mediated AP-2–depleted cells are capable of forming clathrin-coated pits that are competent for the internalization of the EGFR and an LDLR chimera, but defective in TfnR endocytosis (Motley et al., 2003). Thus, we conclude that the functional sequestration of AP-2 by AAK1 overexpression demonstrates an unexpected cargo-selective requirement for this coat constituent in CME.


Differential requirements for AP-2 in clathrin-mediated endocytosis.

Conner SD, Schmid SL - J. Cell Biol. (2003)

AP-2–deficient clathrin-coated pits are functional for EGF uptake. (A) WT AAK1-infected tTA HeLa cells (cultured as described in the legend to Fig. 3 C) show no defect in the recruitment of clathrin to the plasma membrane or its ability to generate coated pits, as observed by immunolocalization using the mAb X22. (B) EGF internalization was tested in tTA HeLa cells infected with adenoviruses expressing either tTA, WT, or K74A AAK1, or K44A dynamin-1 adenoviruses, as indicated in the legend. Protein expression levels were tested by immunoblot analysis (inset) using pAbs against either the ΔAID AAK1 fragment (lanes 1–3) or against dynamin (lanes 1 and 4). (C) tTA HeLa cells, cultured on coverslips and infected with the indicated adenoviruses, were tested for their ability to internalize rhodamine-conjugated Tfn and Alexa-488–conjugated EGF, simultaneously (Molecular Probes). Cells were incubated in the presence of 2 ng/ml EGF and 4 μg/ml Tfn for 15 min at 37°C. Cells were then transferred to ice, washed to remove unbound ligand, fixed with acetone, and visualized by epifluorescence as described in the legend to Fig. 3. A significant decrease in Tfn accumulation in the endosome is observed in WT AAK1–infected cells relative to AID AAK1– or control-infected cells (arrows), whereas EGF uptake is unaffected.
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fig4: AP-2–deficient clathrin-coated pits are functional for EGF uptake. (A) WT AAK1-infected tTA HeLa cells (cultured as described in the legend to Fig. 3 C) show no defect in the recruitment of clathrin to the plasma membrane or its ability to generate coated pits, as observed by immunolocalization using the mAb X22. (B) EGF internalization was tested in tTA HeLa cells infected with adenoviruses expressing either tTA, WT, or K74A AAK1, or K44A dynamin-1 adenoviruses, as indicated in the legend. Protein expression levels were tested by immunoblot analysis (inset) using pAbs against either the ΔAID AAK1 fragment (lanes 1–3) or against dynamin (lanes 1 and 4). (C) tTA HeLa cells, cultured on coverslips and infected with the indicated adenoviruses, were tested for their ability to internalize rhodamine-conjugated Tfn and Alexa-488–conjugated EGF, simultaneously (Molecular Probes). Cells were incubated in the presence of 2 ng/ml EGF and 4 μg/ml Tfn for 15 min at 37°C. Cells were then transferred to ice, washed to remove unbound ligand, fixed with acetone, and visualized by epifluorescence as described in the legend to Fig. 3. A significant decrease in Tfn accumulation in the endosome is observed in WT AAK1–infected cells relative to AID AAK1– or control-infected cells (arrows), whereas EGF uptake is unaffected.
Mentions: The μ2 subunit of AP-2 specifically recognizes tyrosine-based internalization motifs, whereas other AP-2 subunits are believed to function in endocytosis by directing clathrin assembly into curved lattices and by recruiting other essential cofactors to the coated pit (Kirchhausen, 1999). Therefore, we expected that clathrin-coated pit assembly would also be disrupted in cells overexpressing inhibitory AAK1 constructs that functionally sequester AP-2. Surprisingly, clathrin recruitment into coated pits was not altered in WT AAK1–overexpressing cells relative to controls (Fig. 4 A). Previous studies have established that EGF and TfnRs are internalized in the same coated pits (Lamaze et al., 1993). We therefore asked if AAK1 overexpression had any effect on EGF uptake. Surprisingly, neither WT nor K74A AAK1 overexpression had any effect on the internalization of EGF compared with controls (Fig. 4 , B and C). High concentrations of EGF are known to saturate the clathrin-mediated pathway for EGFR endocytosis (Jiang and Sorkin, 2003); therefore, care was taken to use low concentrations (2 ng/ml) of 125I-labeled EGF for these assays. As an additional control for CME, cells infected with recombinant K44A dynamin-1 adenovirus showed the expected EGF internalization defect (Damke et al., 1994). We cannot rule out that the small amounts of AP-2 remaining at the cell surface are selectively associated with coated pits engaged in EGF uptake. However, our results are completely consistent with recent findings reporting that siRNA-mediated AP-2–depleted cells are capable of forming clathrin-coated pits that are competent for the internalization of the EGFR and an LDLR chimera, but defective in TfnR endocytosis (Motley et al., 2003). Thus, we conclude that the functional sequestration of AP-2 by AAK1 overexpression demonstrates an unexpected cargo-selective requirement for this coat constituent in CME.

Bottom Line: Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit.Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo.Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2.

View Article: PubMed Central - PubMed

Affiliation: The Scripps Research Institute, La Jolla, CA 92037, USA.

ABSTRACT
AP-2 complexes are key components in clathrin-mediated endocytosis (CME). They trigger clathrin assembly, interact directly with cargo molecules, and recruit a number of endocytic accessory factors. Adaptor-associated kinase (AAK1), an AP-2 binding partner, modulates AP-2 function by phosphorylating its mu2 subunit. Here, we examined the effects of adenoviral-mediated overexpression of WT AAK1, kinase-dead, and truncation mutants in HeLa cells, and show that AAK1 also regulates AP-2 function in vivo. WT AAK1 overexpression selectively blocks transferrin (Tfn) receptor and LRP endocytosis. Inhibition was kinase independent, but required the full-length AAK1 as truncation mutants were not inhibitory. Although changes in mu2 phosphorylation were not detected, AAK1 overexpression significantly decreased the phosphorylation of large adaptin subunits and the normally punctate AP-2 distribution was dispersed, suggesting that AAK1 overexpression inhibited Tfn endocytosis by functionally sequestering AP-2. Surprisingly, clathrin distribution and EGF uptake were unaffected by AAK1 overexpression. Thus, AP-2 may not be stoichiometrically required for coat assembly, and may have a more cargo-selective function in CME than previously thought.

Show MeSH
Related in: MedlinePlus