"Together, these data suggest that AAK1 corresponds to the endogenous kinase responsible for phosphorylation of μ2 at Thr-156."

"Thus, our in vitro experiments demonstrate that AP2 affinity for sorting signals can be significantly regulated by AAK1-mediated phosphorylation of μ2."

"Thus, these results demonstrate that AAK1 interacts directly with α-adaptin in vitro."

"These results provide strong evidence that μ is a specific target of AAK1 phosphorylation in CCVs and suggests that the observed phosphorylation of μ can be attributed to the presence of endogenous AAK1 in these samples (Fig. 4)."

"Collectively, these data provide functional evidence that AAK1 plays a role in AP2-stimulated endocytosis at an early step by specifically phosphorylating the μ subunit of the AP complex."

"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."

"These data establish that AAK1 can bind to and disrupt the function of AP-2 complexes in vivo."

"Transfection of two different siRNAs that specifically target AAK1 reduced AAK1 expression by ∼80% in either A549 or HeLa cells. However, in neither case did we observe an alteration in Tfn internalization, AP-2 distribution, or μ2 phosphorylation (Fig. S2, available at http://www.jcb.org/cgi/content/full/jcb.200304069/DC1)."

"Here, we report that AAK1 is an atypical kinase that is rate limited by its stable association with AP2 and that clathrin stimulates micro2 phosphorylation by AAK1."

"Importantly, incubation of AAK1 with clathrin cages resulted in even greater stimulation when compared to that of unassembled clathrin triskelia."

"Here we have identified a long form of AAK1 (AAK1L) that contains an extended C-terminus that encodes an additional clathrin-binding domain (CBD2) consisting of multiple low-affinity interaction motifs. Protein interaction studies demonstrate that AAK1L CBD2 directly binds clathrin."

"However, in vitro kinase assays reveal little difference between AAK1 isoforms in their basal or clathrin-stimulated kinase activity toward the AP-2 micro2 subunit."

"Surprisingly, CBD2 overexpression or AAK1 depletion by RNA interference significantly impairs transferrin recycling from the early/sorting endosome."

"We find that AAK1 redistributes Numb to perinuclear endosomes when overexpressed, while kinase depletion causes Numb to accumulate at the plasma membrane."

"Overexpression of a Numb point mutant (T102A) that lacks the AAK1 phosphorylation site potently disrupts transferrin and low-density lipoprotein internalization but does not impact EGF uptake."

"We show here that AAK1, the adaptor-associated kinase 1, directly interacts with the membrane-tethered active form of Notch released by metalloprotease cleavage."

"Active AAK1 acts upstream of the γ-secretase cleavage by stabilizing both the membrane-tethered activated form of Notch and its monoubiquitinated counterpart."

"Moreover, transfected AAK1 increases the localization of activated Notch to Rab5-positive endocytic vesicles, while AAK1 depletion or overexpression of Numb, an inhibitor of the pathway, interferes with this localization."

"Utilizing a split-ubiquitin membrane yeast two-hybrid system that was developed to identify interacting partners of full-length integral membrane proteins, we identified 43 novel interactors that primarily implicate ATP13A2 in cellular processes such as endoplasmic reticulum (ER) translocation, ER-to-Golgi trafficking and vesicular transport and fusion."

"We discovered that two early endosome factors, AAK1L and EHD3, are critical for αvβ3-integrin-mediated cell adhesion in HeLa cells."

"siRNA-mediated depletion of either factor delays short-loop β3 integrin recycling from the early endosome back to the cell surface."

"Moreover, structure-function analysis reveals that AAK1L kinase activity, as well as its C-terminal domain, is essential for cell adhesion maintenance."

"The structure of AAK1 was solved in complex with a small-molecule inhibitor to 1.95 Å resolution in space group P212121 with two AAK1 kinase domains per asymmetric unit (chains A and B) (Table 1)."

"A 17-mer synthetic peptide corresponding to the AAK1/BIKE phosphorylation site on the medium subunit of AP2 was used to test kinase activity. Phosphorylation of the substrate in the presence of AAK1 was confirmed by mass spectrometry (Figure S1)."

"Similarly, in structures of BIKE and AAK1 there is alignment of the R spine in the absence of phosphorylation, suggesting that NAK family members are constitutively active kinases (Figure 2C)."

"Together, these data establish that AAK1 negatively regulates WNT signaling in cells derived from multiple tissue types."

"Together, these results suggest that AAK1 inhibits WNT signaling by inducing CME endocytosis and lysosomal degradation of LRP6."

"The WNT3A-induced phosphorylation of AP2M1 required AAK1 expression and activity."

"Our results showed that AAK1 was more potent than LRRK2 in phosphorylating AP2M1 in vitro."

"We found that AAK1 increased the amount of endogenous AP2M1 phosphorylation in both LRRK2 WT and KO cells (Fig. 3, E and F), suggesting that AAK1 can phosphorylate AP2M1 independently of LRRK2."

"Notably, we found that unlike LRRK2, the expression of AAK1 was relatively low in mouse brain tissue (Fig. 3, I and J). In contrast, AAK1 was highly expressed in the mouse thyroid gland where LRRK2 expression was relatively low."

"The level of Ser/Thr phosphorylation of AAK1 protein increased significantly in the presence of recombinant active PKCβII kinase, but was blocked in the addition of λ-phosphatase (Fig. 3E, F)."

"Mechanistically, activated AAK1 phosphorylates AP2M1, which facilitates the recruitment of clathrin to mediate the endocytosis of TFR1, increasing the levels of both cellular total iron and ferrous iron and thereby promoting ferroptosis."

"To further confirm the role of AAK1 during the process of ferroptosis in vivo, we performed xenograft tumor model by inoculating AAK1-knockout MDA-MB-231 cells with or without transfecting plasmids of wild-type or S670/T674Ala mutant AAK1 into nude mice. These nude mice were treated with IKE (a lipid-soluble form of erastin for animal experiment) or DMSO as control. As expected, knockout of AAK1 significantly inhibited ferroptosis through resistance of lipid peroxidation induced by IKE and promoted tumor growth (Fig. 6G–J)."

"Here, using motif-guided in silico, biochemical, and phosphoproteomic screens, we identify PDLIM5 and Talin1 as direct AAK1/BMP2K substrates."

"AAK1 deletion or siRNA-mediated knockdown reproducibly impaired RPE migration by ~25% (Fig. 3d, e and Supplementary Fig. 1h), whereas BMP2K loss had no measurable effect, revealing a specific and non-redundant role for AAK1 in cell motility."

"Furthermore, our global phosphoproteome profiling of RPE cells failed to detect phosphorylation of the previously proposed substrates Dab2 T109, Arh T104, or NUMB T102. This conclusion is supported by both our in vitro kinase assays and the absence of phosphorylation evidence in high-throughput databases, such as PhosphoSitePlus (Fig. 1h, i), which suggests that Dab2 T109, Arh T104, and NUMB T102 are unlikely to be physiological targets of AAK1 or BMP2K."