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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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Full-length AAK1 inhibits Tfn uptake in vivo. tTA-HeLa cells were infected with either control adenovirus encoding the tTA transcription activator or tetracycline-regulatable adenoviruses encoding either full-length WT AAK1 or kinase-inactive mutants (K74A or D176A) (A), or AAK1 fragments (B), as indicated in the legend, and tested for Tfn endocytosis as described in the Materials and methods. Protein expression levels were tested by immunoblot analysis (insets), using pAbs against ΔAID AAK1 and/or the AID AAK1 fragment. Each time point represents the average of three independent experiments ± the standard deviation. (C) The observed inhibition is concentration dependent. Cells were infected with WT AAK1 adenovirus in the presence of increasing concentrations of tetracycline to titrate AAK1 expression, and Tfn endocytosis was assessed as described here.
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fig2: Full-length AAK1 inhibits Tfn uptake in vivo. tTA-HeLa cells were infected with either control adenovirus encoding the tTA transcription activator or tetracycline-regulatable adenoviruses encoding either full-length WT AAK1 or kinase-inactive mutants (K74A or D176A) (A), or AAK1 fragments (B), as indicated in the legend, and tested for Tfn endocytosis as described in the Materials and methods. Protein expression levels were tested by immunoblot analysis (insets), using pAbs against ΔAID AAK1 and/or the AID AAK1 fragment. Each time point represents the average of three independent experiments ± the standard deviation. (C) The observed inhibition is concentration dependent. Cells were infected with WT AAK1 adenovirus in the presence of increasing concentrations of tetracycline to titrate AAK1 expression, and Tfn endocytosis was assessed as described here.

Mentions: We next infected tetracycline transactivator (tTA) HeLa cells with adenoviruses encoding various AAK1 constructs and assayed for their ability to internalize biotinylated Tfn. Cells overexpressing WT AAK1 showed a significant, concentration-dependent inhibition in Tfn endocytosis compared with controls (Fig. 2, A and C), a result consistent with in vitro observations (Conner and Schmid, 2002). Although somewhat less potent, inhibition was also observed in cells overexpressing either of the kinase-inactive AAK1 mutants. This is also consistent with the weaker, but observable inhibition of FSBA-treated AAK1 on endocytosis in perforated cells (Conner and Schmid, 2002). Interestingly, neither overexpression of the kinase-active ΔAID, nor the QPA or AID fragments of AAK1, which we expected to effectively compete for AAK1-interacting partners and thus perturb AAK1 function, significantly affected Tfn endocytosis (Fig. 2 B). From these observations, we conclude that the internalization defects seen with full-length AAK1 constructs do not simply reflect rampant kinase activity, a sequestration of AAK1-interacting partners, or interference with other AP-2 partners that interact through the ear domain of α adaptin.


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

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

Full-length AAK1 inhibits Tfn uptake in vivo. tTA-HeLa cells were infected with either control adenovirus encoding the tTA transcription activator or tetracycline-regulatable adenoviruses encoding either full-length WT AAK1 or kinase-inactive mutants (K74A or D176A) (A), or AAK1 fragments (B), as indicated in the legend, and tested for Tfn endocytosis as described in the Materials and methods. Protein expression levels were tested by immunoblot analysis (insets), using pAbs against ΔAID AAK1 and/or the AID AAK1 fragment. Each time point represents the average of three independent experiments ± the standard deviation. (C) The observed inhibition is concentration dependent. Cells were infected with WT AAK1 adenovirus in the presence of increasing concentrations of tetracycline to titrate AAK1 expression, and Tfn endocytosis was assessed as described here.
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Related In: Results  -  Collection

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fig2: Full-length AAK1 inhibits Tfn uptake in vivo. tTA-HeLa cells were infected with either control adenovirus encoding the tTA transcription activator or tetracycline-regulatable adenoviruses encoding either full-length WT AAK1 or kinase-inactive mutants (K74A or D176A) (A), or AAK1 fragments (B), as indicated in the legend, and tested for Tfn endocytosis as described in the Materials and methods. Protein expression levels were tested by immunoblot analysis (insets), using pAbs against ΔAID AAK1 and/or the AID AAK1 fragment. Each time point represents the average of three independent experiments ± the standard deviation. (C) The observed inhibition is concentration dependent. Cells were infected with WT AAK1 adenovirus in the presence of increasing concentrations of tetracycline to titrate AAK1 expression, and Tfn endocytosis was assessed as described here.
Mentions: We next infected tetracycline transactivator (tTA) HeLa cells with adenoviruses encoding various AAK1 constructs and assayed for their ability to internalize biotinylated Tfn. Cells overexpressing WT AAK1 showed a significant, concentration-dependent inhibition in Tfn endocytosis compared with controls (Fig. 2, A and C), a result consistent with in vitro observations (Conner and Schmid, 2002). Although somewhat less potent, inhibition was also observed in cells overexpressing either of the kinase-inactive AAK1 mutants. This is also consistent with the weaker, but observable inhibition of FSBA-treated AAK1 on endocytosis in perforated cells (Conner and Schmid, 2002). Interestingly, neither overexpression of the kinase-active ΔAID, nor the QPA or AID fragments of AAK1, which we expected to effectively compete for AAK1-interacting partners and thus perturb AAK1 function, significantly affected Tfn endocytosis (Fig. 2 B). From these observations, we conclude that the internalization defects seen with full-length AAK1 constructs do not simply reflect rampant kinase activity, a sequestration of AAK1-interacting partners, or interference with other AP-2 partners that interact through the ear domain of α adaptin.

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