Novel phosphorylation substrates reveal a spatially regulated role for AAK1 in cell migration

NAKs (Numb-associated kinases) are a relatively unexplored kinase family that interacts with the endocytic machinery. Among NAK members, AAK1 has been linked to disorders such as neuropathic pain, schizophrenia, and Alzheimer's disease. Our goal is to better understand the cellular pathways regulated by AAK1 and its paralog BMP2K, as their only known role until now has been in endocytosis.

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5488 - Characterization of AAK1/BMP2K Phosphorylation Substrates Using PRM Acquisition in RPE Cells
Using the LFQ approach with PRM acquisition for preselected phosphopeptides containing the [L/I]XXQXTG motif, we analyzed whole-cell samples from hTERT RPE cells with genetic and pharmacological perturbations of these kinases. Our findings reveal that the double knockout and inhibition of AAK1 and BMP2K significantly reduces the abundance of PDLIM5 and TLN1 phosphopeptides, ILAQI{pT}GTEHLK and QQL{pT}GHSK respectively, identifying them as bona fide substrates of AAK1/BMP2K.

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5565 - AAK1 Overexpression in RPE Cells increases Phosphorylation of PDLIM5 at ILAQI{pT}GTEHLK
Using the LFQ approach with PRM acquisition, we analyzed the PDLIM5 phosphopeptide ILAQI{pT}GTEHLK in samples where PDLIM5 was immunoprecipitated from hTERT RPE cells with AAK1 overexpression as well as wild type cells. Our results show that AAK1 overexpression significantly increases the abundance of this PDLIM5 phosphopeptide compared to wild-type samples. These findings confirm PDLIM5 as a substrate of AAK1.

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5759 - Targeted Phosphoproteomics Uncovers PDLIM5 and TLN1 as AAK1/BMP2K substrates in RPE Cells
Using the LFQ approach with PRM acquisition, we analyzed samples with PDLIM5 or TLN1 immunoprecipitated from hTERT RPE cells with genetic and pharmacological perturbations of AAK1 and BMP2K. We quantified the abundance of the PDLIM5 and TLN1 phosphopeptides ILAQI{pT}GTEHLK and QQL{pT}GHSK respectively, finding that both the double knockout and inhibition of these kinases significantly decrease the abundance of the PDLIM5 phosphopeptide and the double knockout significantly decreases the amount of the TLN1 phosphopeptide. These findings identify PDLIM5 and TLN1 as substrates of AAK1 and BMP2K.

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6045, 6138 - In Vitro Phosphorylation Screening of possible AAK1 and BMP2K Substrate Peptides
Using an NADH-coupled in vitro assay, we tested peptides preselected from an in silico screen to determine their phosphorylation by AAK1 and/or BMP2K. The assay products were subsequently analyzed by mass spectrometry, with phosphorylation at the target residues verified through Mascot analysis and confirmed by manual inspection in Skyline.