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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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AAK1 globally disrupts AP-2 function. (A) Adenovirally-infected tTA HeLa cells overexpressing WT AAK1, K74A AAK1, or the tTA were labeled in vivo with 32P-orthophosphate. AP-2 was then immunoprecipitated with the mAb AP.6 and analyzed by SDS-PAGE (top). Immunoblot (bottom) with antibodies specific for μ2 (provided by J. Bonifacino, National Institutes of Health, Bethesda, MD) indicates equal loading. (B) Quantitation of phosphorylated large adaptin subunits from whole cell lysates relative to the tTA control. Data shown are representative of three independent experiments. (C) tTA HeLa cells, cultured in the absence of G418, were infected with the indicated AAK1 or dynamin adenovirus constructs. All cells are virally infected, but only those cells that retain tTA express the adenovirus-encoded constructs. Infected cells were fixed with ice-cold acetone and methanol extracted before further processing for immunolocalization of AAK1 using pAbs against either the ΔAID or AID fragment and the mAb AP.6 that recognizes the α-adaptin subunit of AP-2. Samples were visualized by epifluorescence microscopy using a Zeiss Axiophot with an attached Zeiss Axiocam. (D) The distribution of AP-2 in particulate (P) and soluble (S) fractions, obtained as previously described (Damke et al., 1994), was tested in adenovirus-infected cells by immunoblot analysis, using the mAb 100/2 (Sigma-Aldrich).
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fig3: AAK1 globally disrupts AP-2 function. (A) Adenovirally-infected tTA HeLa cells overexpressing WT AAK1, K74A AAK1, or the tTA were labeled in vivo with 32P-orthophosphate. AP-2 was then immunoprecipitated with the mAb AP.6 and analyzed by SDS-PAGE (top). Immunoblot (bottom) with antibodies specific for μ2 (provided by J. Bonifacino, National Institutes of Health, Bethesda, MD) indicates equal loading. (B) Quantitation of phosphorylated large adaptin subunits from whole cell lysates relative to the tTA control. Data shown are representative of three independent experiments. (C) tTA HeLa cells, cultured in the absence of G418, were infected with the indicated AAK1 or dynamin adenovirus constructs. All cells are virally infected, but only those cells that retain tTA express the adenovirus-encoded constructs. Infected cells were fixed with ice-cold acetone and methanol extracted before further processing for immunolocalization of AAK1 using pAbs against either the ΔAID or AID fragment and the mAb AP.6 that recognizes the α-adaptin subunit of AP-2. Samples were visualized by epifluorescence microscopy using a Zeiss Axiophot with an attached Zeiss Axiocam. (D) The distribution of AP-2 in particulate (P) and soluble (S) fractions, obtained as previously described (Damke et al., 1994), was tested in adenovirus-infected cells by immunoblot analysis, using the mAb 100/2 (Sigma-Aldrich).

Mentions: In vitro kinase assays did not reveal any major AAK1 targets other than μ2 in either cytosolic or membrane fractions (Conner and Schmid, 2002). Moreover, μ2 phosphorylation is known to be required for endocytosis in vivo (Olusanya et al., 2001). Thus, we expected that overexpression of full-length AAK1 constructs inhibited AP-2 function by shifting the balance of μ2 into a phosphorylated (WT AAK1) or dephosphorylated (K74A or D176A AAK1) state. However, immunoprecipitation of AP-2 complexes from whole cell lysates, following in vivo labeling, did not show any significant alteration in μ2 phosphorylation in cells overexpressing either WT or kinase-dead AAK1 (Fig. 3 A). Although we cannot rule out the existence of a specifically localized subpopulation of phosphorylated μ2, these data suggest that μ2 phosphorylation activity of AAK1 in vivo is tightly regulated. Unexpectedly, a significant decrease in phosphorylation of the large AP-2 subunits was observed. Thus, rather than altering the μ2 phosphorylation state, the observed receptor internalization block appears to result from the kinase activity–independent binding of full-length AAK1 to AP-2, which exerts a more global effect on AP-2 function.


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

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

AAK1 globally disrupts AP-2 function. (A) Adenovirally-infected tTA HeLa cells overexpressing WT AAK1, K74A AAK1, or the tTA were labeled in vivo with 32P-orthophosphate. AP-2 was then immunoprecipitated with the mAb AP.6 and analyzed by SDS-PAGE (top). Immunoblot (bottom) with antibodies specific for μ2 (provided by J. Bonifacino, National Institutes of Health, Bethesda, MD) indicates equal loading. (B) Quantitation of phosphorylated large adaptin subunits from whole cell lysates relative to the tTA control. Data shown are representative of three independent experiments. (C) tTA HeLa cells, cultured in the absence of G418, were infected with the indicated AAK1 or dynamin adenovirus constructs. All cells are virally infected, but only those cells that retain tTA express the adenovirus-encoded constructs. Infected cells were fixed with ice-cold acetone and methanol extracted before further processing for immunolocalization of AAK1 using pAbs against either the ΔAID or AID fragment and the mAb AP.6 that recognizes the α-adaptin subunit of AP-2. Samples were visualized by epifluorescence microscopy using a Zeiss Axiophot with an attached Zeiss Axiocam. (D) The distribution of AP-2 in particulate (P) and soluble (S) fractions, obtained as previously described (Damke et al., 1994), was tested in adenovirus-infected cells by immunoblot analysis, using the mAb 100/2 (Sigma-Aldrich).
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fig3: AAK1 globally disrupts AP-2 function. (A) Adenovirally-infected tTA HeLa cells overexpressing WT AAK1, K74A AAK1, or the tTA were labeled in vivo with 32P-orthophosphate. AP-2 was then immunoprecipitated with the mAb AP.6 and analyzed by SDS-PAGE (top). Immunoblot (bottom) with antibodies specific for μ2 (provided by J. Bonifacino, National Institutes of Health, Bethesda, MD) indicates equal loading. (B) Quantitation of phosphorylated large adaptin subunits from whole cell lysates relative to the tTA control. Data shown are representative of three independent experiments. (C) tTA HeLa cells, cultured in the absence of G418, were infected with the indicated AAK1 or dynamin adenovirus constructs. All cells are virally infected, but only those cells that retain tTA express the adenovirus-encoded constructs. Infected cells were fixed with ice-cold acetone and methanol extracted before further processing for immunolocalization of AAK1 using pAbs against either the ΔAID or AID fragment and the mAb AP.6 that recognizes the α-adaptin subunit of AP-2. Samples were visualized by epifluorescence microscopy using a Zeiss Axiophot with an attached Zeiss Axiocam. (D) The distribution of AP-2 in particulate (P) and soluble (S) fractions, obtained as previously described (Damke et al., 1994), was tested in adenovirus-infected cells by immunoblot analysis, using the mAb 100/2 (Sigma-Aldrich).
Mentions: In vitro kinase assays did not reveal any major AAK1 targets other than μ2 in either cytosolic or membrane fractions (Conner and Schmid, 2002). Moreover, μ2 phosphorylation is known to be required for endocytosis in vivo (Olusanya et al., 2001). Thus, we expected that overexpression of full-length AAK1 constructs inhibited AP-2 function by shifting the balance of μ2 into a phosphorylated (WT AAK1) or dephosphorylated (K74A or D176A AAK1) state. However, immunoprecipitation of AP-2 complexes from whole cell lysates, following in vivo labeling, did not show any significant alteration in μ2 phosphorylation in cells overexpressing either WT or kinase-dead AAK1 (Fig. 3 A). Although we cannot rule out the existence of a specifically localized subpopulation of phosphorylated μ2, these data suggest that μ2 phosphorylation activity of AAK1 in vivo is tightly regulated. Unexpectedly, a significant decrease in phosphorylation of the large AP-2 subunits was observed. Thus, rather than altering the μ2 phosphorylation state, the observed receptor internalization block appears to result from the kinase activity–independent binding of full-length AAK1 to AP-2, which exerts a more global effect on AP-2 function.

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