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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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Structural requirements for AAK1-mediated μ2 phosphorylation. (A) Diagram illustrating the AAK1 constructs used in this study. Baculovirus constructs were GST-tagged at the COOH terminus, whereas adenovirus constructs were HA-tagged at the NH2 terminus (see supplemental methods, available at http://www.jcb.org/cgi/content/full/jcb.200304069/DC1). (B) FSBA-inactivated APs (5.2 μg) were incubated with AAK1–GST fusion proteins, as indicated, in the presence of [γ32P]ATP and phosphorylated protein detected by SDS-PAGE and autoradiography. Assays were performed with 0.25 μM of WT, K74A, or D176A AAK1 and 0.65 μM (2.5×) or 2.5 μM (10×) of the ΔAID mutant. (C) The indicated AAK1–GST fusion proteins were immobilized on glutathione–agarose beads at either ∼0.25 mg/ml (1×) or ∼2.5 mg/ml (10×) and incubated with isolated APs. Bound AP-2 was detected by immunoblot analysis.
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fig1: Structural requirements for AAK1-mediated μ2 phosphorylation. (A) Diagram illustrating the AAK1 constructs used in this study. Baculovirus constructs were GST-tagged at the COOH terminus, whereas adenovirus constructs were HA-tagged at the NH2 terminus (see supplemental methods, available at http://www.jcb.org/cgi/content/full/jcb.200304069/DC1). (B) FSBA-inactivated APs (5.2 μg) were incubated with AAK1–GST fusion proteins, as indicated, in the presence of [γ32P]ATP and phosphorylated protein detected by SDS-PAGE and autoradiography. Assays were performed with 0.25 μM of WT, K74A, or D176A AAK1 and 0.65 μM (2.5×) or 2.5 μM (10×) of the ΔAID mutant. (C) The indicated AAK1–GST fusion proteins were immobilized on glutathione–agarose beads at either ∼0.25 mg/ml (1×) or ∼2.5 mg/ml (10×) and incubated with isolated APs. Bound AP-2 was detected by immunoblot analysis.

Mentions: To test the in vivo role of AAK1 in regulating AP-2 function, we generated recombinant tetracycline-regulatable adenoviruses encoding AAK1 constructs postulated to compete for endogenous AAK1 function (Fig. 1 A). Identical constructs for baculovirus protein expression were also generated for biochemical analysis to allow for correlation of in vivo and in vitro observations. Endogenous kinase(s), including AAK1, that are known to cofractionate with AP-2 (Conner and Schmid, 2002; Korolchuk and Banting, 2002) were first inactivated by pretreatment with FSBA, an ATP analogue and irreversible kinase inhibitor (Fig. 1 B; Olusanya et al., 2001). Addition of WT AAK1 to FSBA-inactivated AP-2 resulted in efficient μ2 phosphorylation when incubated in the presence of [γ32P]ATP (Fig. 1 B; Conner and Schmid, 2002). As expected, point mutations of conserved residues within the kinase domain, either K74A or D176A, predicted to disrupt nucleotide binding and catalysis, respectively, severely inhibited AAK1 activity for either phosphorylation of μ2 or autophosphorylation. A truncated AAK1 construct (ΔAID) that lacks the α-adaptin–interacting domain (AID) was efficiently autophosphorylated, thus ΔAID AAK1 is a fully active kinase. However, 10-fold more ΔAID was required to phosphorylate μ2 (Fig. 1 B). Thus, although the majority of AP-2 interaction is supported by the AID, other lower affinity AP-2 binding domains must exist. Indeed, GST fusion protein pull-downs demonstrate that most AP-2 binding is supported by the AID fragment (Fig. 1 C); however, AP-2 binding by both the ΔAID and QPA fragments was detected when ∼10-fold more AAK1 fragment–GST fusion protein was used (Fig. 1 C). We conclude that efficient recruitment of AAK1 to AP-2 and its phosphorylation of μ2 requires the AID and that each of the individual domains and truncated mutants retain their expected activities.


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

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

Structural requirements for AAK1-mediated μ2 phosphorylation. (A) Diagram illustrating the AAK1 constructs used in this study. Baculovirus constructs were GST-tagged at the COOH terminus, whereas adenovirus constructs were HA-tagged at the NH2 terminus (see supplemental methods, available at http://www.jcb.org/cgi/content/full/jcb.200304069/DC1). (B) FSBA-inactivated APs (5.2 μg) were incubated with AAK1–GST fusion proteins, as indicated, in the presence of [γ32P]ATP and phosphorylated protein detected by SDS-PAGE and autoradiography. Assays were performed with 0.25 μM of WT, K74A, or D176A AAK1 and 0.65 μM (2.5×) or 2.5 μM (10×) of the ΔAID mutant. (C) The indicated AAK1–GST fusion proteins were immobilized on glutathione–agarose beads at either ∼0.25 mg/ml (1×) or ∼2.5 mg/ml (10×) and incubated with isolated APs. Bound AP-2 was detected by immunoblot analysis.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172816&req=5

fig1: Structural requirements for AAK1-mediated μ2 phosphorylation. (A) Diagram illustrating the AAK1 constructs used in this study. Baculovirus constructs were GST-tagged at the COOH terminus, whereas adenovirus constructs were HA-tagged at the NH2 terminus (see supplemental methods, available at http://www.jcb.org/cgi/content/full/jcb.200304069/DC1). (B) FSBA-inactivated APs (5.2 μg) were incubated with AAK1–GST fusion proteins, as indicated, in the presence of [γ32P]ATP and phosphorylated protein detected by SDS-PAGE and autoradiography. Assays were performed with 0.25 μM of WT, K74A, or D176A AAK1 and 0.65 μM (2.5×) or 2.5 μM (10×) of the ΔAID mutant. (C) The indicated AAK1–GST fusion proteins were immobilized on glutathione–agarose beads at either ∼0.25 mg/ml (1×) or ∼2.5 mg/ml (10×) and incubated with isolated APs. Bound AP-2 was detected by immunoblot analysis.
Mentions: To test the in vivo role of AAK1 in regulating AP-2 function, we generated recombinant tetracycline-regulatable adenoviruses encoding AAK1 constructs postulated to compete for endogenous AAK1 function (Fig. 1 A). Identical constructs for baculovirus protein expression were also generated for biochemical analysis to allow for correlation of in vivo and in vitro observations. Endogenous kinase(s), including AAK1, that are known to cofractionate with AP-2 (Conner and Schmid, 2002; Korolchuk and Banting, 2002) were first inactivated by pretreatment with FSBA, an ATP analogue and irreversible kinase inhibitor (Fig. 1 B; Olusanya et al., 2001). Addition of WT AAK1 to FSBA-inactivated AP-2 resulted in efficient μ2 phosphorylation when incubated in the presence of [γ32P]ATP (Fig. 1 B; Conner and Schmid, 2002). As expected, point mutations of conserved residues within the kinase domain, either K74A or D176A, predicted to disrupt nucleotide binding and catalysis, respectively, severely inhibited AAK1 activity for either phosphorylation of μ2 or autophosphorylation. A truncated AAK1 construct (ΔAID) that lacks the α-adaptin–interacting domain (AID) was efficiently autophosphorylated, thus ΔAID AAK1 is a fully active kinase. However, 10-fold more ΔAID was required to phosphorylate μ2 (Fig. 1 B). Thus, although the majority of AP-2 interaction is supported by the AID, other lower affinity AP-2 binding domains must exist. Indeed, GST fusion protein pull-downs demonstrate that most AP-2 binding is supported by the AID fragment (Fig. 1 C); however, AP-2 binding by both the ΔAID and QPA fragments was detected when ∼10-fold more AAK1 fragment–GST fusion protein was used (Fig. 1 C). We conclude that efficient recruitment of AAK1 to AP-2 and its phosphorylation of μ2 requires the AID and that each of the individual domains and truncated mutants retain their expected activities.

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