Report |
Address correspondence to Stefan Höning, Institute for Biochemistry II, University of Göttingen, Heinrich-Duker-Weg 12, Göttingen 37073, Germany. Tel.: 49-551-392127. Fax.: 49-551-395979. E-mail: shoenin{at}gwdg.de
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Abstract |
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Key Words: clathrin; endocytosis; receptor; trafficking; coated vesicle
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Introduction |
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Protein kinases have long been known to associate with CCVs (Campbell et al., 1984; Pauloin et al., 1984; Bar-Zvi and Branton, 1986), and phosphorylation plays a role in regulating protein interactions in receptor-mediated endocytosis (Slepnev et al., 1998; Cousin and Robinson, 2001). For example, AP2 must be dephosphorylated to mediate clathrin assembly (Wilde and Brodsky, 1996). We recently demonstrated that phosphorylation of AP2 by an endogenous kinase(s) enhances binding to the internalization motifs of membrane proteins (Fingerhut et al., 2001). However, neither the identity of the kinase(s) involved, nor which of the four AP2 subunits required phosphorylation to mediate high affinity binding to membrane protein sorting signals was known. Here we show that µ2 phosphorylation accounts for the enhanced affinity for sorting motifs, and we identify AAK1, a newly discovered adaptor-associated kinase (Conner and Schmid, 2002) as the responsible kinase.
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Results and discussion |
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To obtain AP2 free of kinase activity, we immunoisolated AP2 from pool 4 using an -adaptinspecific antibody. After immunoisolation, the immunocomplexes were extensively washed using high salt and Tween before incubation with [
32P]ATP. Virtually no phosphorylation of any AP2 subunit was detectable (Fig. 1 C, lane 1), showing that immunoisolated AP2 was free of AP2-phosphorylating kinases. When immunoisolated AP2 was incubated with [
32P]ATP and an aliquot of the HAP-derived pools (Fig. 1 A), the strong phosphorylation of a 50-kD protein corresponding to the AP2 µ2 subunit was detected (Fig. 1 C, lanes 24). The µ2-phosphorylating activity was highest in pools 3 and 4, but was also readily detectable in pool 2. In addition to µ2 phosphorylation, a weak but significant phosphorylation of the AP2 large subunits became apparent. Taken together, these experiments show that kinases that predominantly phosphorylate the µ2 subunit copurify with AP2 but can be separated by immunoadsorption and extensive washing.
AAK1, a µ2-phosphorylating kinase, copurifies with AP2
Two kinases have recently been shown to mediate phosphorylation of the µ2 subunit: GAK/auxilin-2 (Umeda et al., 2000) and the newly discovered AAK1 (Conner and Schmid, 2002). Whereas AAK1, which copurifies with adaptor fractions isolated by gel filtration chromatography, colocalizes to endocytic structures, GAK/auxilin-2 was shown to be predominantly cytosolic, with only a small fraction concentrated in perinuclear structures that colocalize with AP1 (Greener et al., 2000). Recombinant GAK/auxilin-2 can mediate µ2 phosphorylation, however, it is not known whether the kinase copurifies with AP2. We therefore analyzed the distribution of GAK/auxilin-2 and AAK1 during the AP2 purification (Fig. 2)
. Both kinases can be detected in pig brainderived CCVs (Fig. 2, lane 1); however, the two kinases were resolved by further fractionation. After extraction of CCVs with 0.5 M Tris, pH 7.8, most of GAK/auxilin-2 remained associated with the pelleted membranes (Fig. 2, lane 2), whereas 50% of AAK1 was recovered in the supernatant together with extracted coat proteins, including AP2. AAK1 was slightly enriched in the adaptor-containing fractions after gel filtration (lane 4), whereas GAK/auxilin-2 was further depleted from the adaptor-containing pool. After fractionation on HAP (Fig. 1 A), the remaining GAK/auxilin-2 was enriched in AP1-containing fractions (pool 2), but almost depleted from AP2-containing fractions (pool 3 and 4), consistent with its localization to the perinuclear region of cells (Greener et al., 2000). In contrast, AAK1 was detectable in pools 24, with a maximum in pool 4. The amount of 32P incorporated into µ2 correlated with the amount of AAK1 in pools 3 and 4, rather than with the amount of AP2 (Fig. 2 B), suggesting that AAK1 is the kinase that phosphorylates µ2 in pools 3 and 4.
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µ2 phosphorylation by AAK1 mediates high affinity binding of AP2 to sorting signals
As mentioned above, phosphorylation of AP2 increases its affinity for membrane protein sorting signals (Fingerhut et al., 2001); however neither the kinase(s) involved, nor the AP2 subunit(s) whose phosphorylation was responsible for the increase in affinity had been identified. Given that µ2 is known to bind to tyrosine-based sorting signals (Aguilar et al., 1997; Owen and Evans, 1998), we first analyzed whether the selective phosphorylation of µ2 modulates AP2 binding affinity. The kinetics of AP2 binding were determined by surface plasmon resonance using a synthetic peptide representing the tyrosine-based sorting motif of lysosomal acid phosphatase (LAP) immobilized on a biosensor surface. In agreement with previously published data, HAP-purified AP2 (pool 4) effectively bound to the LAP tail (Fig. 4
, sensorgram 1), but its affinity decreased more than fourfold when AP2 was treated with phosphatase before the biosensor experiment (sensorgram 2), suggesting that AP2 in pool 4 was partially phosphorylated. In contrast, AP2 incubation with ATP resulted in an eightfold increase in affinity (Fig. 4, sensorgram 3) as compared with dephosphorylated AP2. This increase in binding affinity was sensitive to the addition of staurosporine (Fig. 4, sensorgram 5). Incubation of AP2 without ATP, but in the presence of staurosporine, did not decrease the binding affinity, as compared with the untreated control (unpublished data), indicating that pool 4 does not contain a µ2-specific phosphatase.
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It is important to note that all biosensor experiments described here were performed in the presence of divalent Mn2+ ions to establish conditions that only allow µ2 phosphorylation, but selectively abolish the phosphorylation of and ß2 (Fig. 1 B). In parallel to the affinity measurements, we verified the selective phosphorylation of µ2 in identical samples under identical conditions, however in the presence of [
32P]ATP (unpublished data). When the same biosensor experiments were performed without the addition of Mn2+, resulting in the weak but significant phosphorylation of
and ß2, we did not observe any difference with the results presented here (unpublished data). Taken together, our in vitro data clearly show that the AAK1-mediated phosphorylation of the AP2 µ subunit alone is sufficient to modulate the affinity of AP2 towards sorting signals, whereas the phosphorylation status of the adaptor large subunits is of no relevance.
Our data is in line with and provide a convincing explanation for the recent findings that phosphorylation of µ2 on Thr-156 is critical for AP2 function in receptor-mediated endocytosis both in vitro and in vivo (Olusanya et al., 2001). Using a perforated A431 cell assay, the authors demonstrated that µ2 phosphorylation was required for AP2-stimulated transferrin sequestration into coated pits, and also showed that overexpression of µ2 (T156A) mutants inhibited receptor-mediated endocytosis. Our results now demonstrate that the inhibition of endocytosis can be attributed to the loss of efficient AP2 binding to the membrane protein sorting signals due to inactivation of the µ2 phosphorylation site Thr-156.
Interestingly, using the same perforated A431 cell assay, AAK1 was shown to inhibit AP2-dependent transferrin sequestration into constricted coated pits and coated vesicles in a kinase activitydependent manner (Conner and Schmid, 2002). Taken together, these data are compatible with the view that continuous endocytosis requires cycles of phosphorylation/dephosphorylation of AP2, allowing the utilization of AP2 for multiple rounds of cargo recruitment. Alternatively, the inhibitory effect of excess AAK1 in this assay could reflect phosphorylation of other as yet unidentified substrates. Further work will be necessary to define the respective roles of AAK1 and GAK and to determine how these kinases are themselves regulated and how they regulate AP2 function and receptor-mediated endocytosis.
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Materials and methods |
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Preparation of AP2
The preparation of CCVs from porcine brain, the Tris extraction of coat proteins from CCVs, the Superose 6 gel filtration during which adaptors are enriched, and the HAP chromatography for the separation of AP2 from AP1 were performed exactly as previously described (Fingerhut et al., 2001). The purity of AP1 and AP2 was analyzed by SDS-PAGE and Western blotting, using specific antibodies for AP1 and AP2. Fractions containing AP2 were pooled and dialyzed into either buffer A (25 mM Hepes, 125 mM KOAc, 10 mM NaCl, 5 mM MgCl2, 1 mM DTT, pH 7.2) or buffer B (100 mM Tris-Cl, 1 mM DTT, pH 7.2), as indicated. In some experiments, buffer A or B was supplemented with 2 mM MnCl2.
Immunoisolation of AP2 was performed by first absorbing the anti-adaptin antibody AP.6 (Alexis Biochemicals Corp.) to protein ASepharose (Sigma-Aldrich) followed by incubation with AP2 for 4 h at 4°C. After immunoisolation, the immune complexes were washed with 0.5 M NaCl in PBS and 0.1% Tween 20 in PBS before they were used for further experiments.
In vitro phosphorylation and dephosphorylation of AP2
AP2 dialyzed into buffer A, was incubated with 1 µl [32P]ATP (3,000 Ci/mmol; Amersham Pharmacia Biotech) for 20 min at room temperature (RT). The 20-µl reaction was stopped by the addition of sample buffer, boiled for 5 min at 95°C, and analyzed by SDS-PAGE, autoradiography, and Western blotting with antibodies against the µ2 subunit. The phosphorylation of immunoisolated AP2 (20 µl) was performed in 50 µl of buffer A or in 50 µl of pooled kinase-containing fractions (Fig. 1). In some experiments, recombinant AAK1, purified as previously described (Conner and Schmid, 2002), was added to AP2 under the same buffer conditions at a molar ratio of 1:20 (AAK1/AP2). The reaction was stopped by washing three times with ice-cold PBS, containing 0.1% Tween 20. Samples were analyzed as above. The kinase inhibitor experiments are described in the legend of Fig. 3. All inhibitors (Alexis Biochemicals Corp.) were used at concentrations recommended to abolish their target kinases. For phosphorylation with unlabeled ATP, AP2 was incubated for 20 min at 37°C in the presence of 2 mM ATP in buffer A or B, with or without Mn2+.
Dephosphorylation of AP2 was performed in buffer A or B with various amounts of calf intestinal alkaline phosphatase (Boehringer) for 20 min at 37°C.
Biosensor-based interaction analysis
The interaction between AP2 and the cytoplasmic tail peptides was analyzed in real time by surface plasmon resonance, using a BIAcore3000 biosensor (BIAcore AB). Immobilization of peptides corresponding to the tail sequence from LAP to the biosensor surface was exactly as described by Fingerhut et al. (2001). The experiments were performed at a flow rate of 20 µl/min, and AP2 was used at concentrations of 50 and 250 nM. Association (12 min) was followed by dissociation (2 min), during which buffer A was perfused. Subsequently, bound AP2 was removed by a short pulse injection (15 s) of 10 mM NaOH containing 0.5% SDS.
The rate constants (ka for association, kd for dissociation, and the equilibrium rate constant KD = kd/ka) for the interaction between the tail peptides and AP2 were calculated as previously described (Höning et al., 1997; Fingerhut et al., 2001).
Online supplemental material
Fig. S1 (available online at http://www.jcb.org/cgi/content/full/jcb.200111068/DC1) shows results from reverse phase HPLC purification of phosphopeptides after tryptic digestion of phosphorylated µ2, which was used together with MALDI/MALDI MS and radiosequencing to map the phosphorylation site.
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Footnotes |
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* Abbreviations used in this paper: AAK, adaptor-associated kinase; CCV, clathrin-coated vesicle; HAP, hydroxyapatite; LAP, lysosomal acid phosphatase; RT, room temperature.
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Acknowledgments |
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This work was supported by grants from the European Union (XCT 960058), the Deutsche Forschungsgemeinschaft (SFB 523/TP A5), the Fonds der Chemischen Industrie, and the National Institutes of Health (R37-MH61345 to S.L. Schmid and GM20632 to S.D. Conner).
Submitted: 19 November 2001
Revised: 15 January 2002
Accepted: 16 January 2002
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References |
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