Thousand and one amino acid kinases 1 (TAOK1), also known as microtubule affinity regulating kinase (MARKK) or Prostate-derived sterile 20-like kinases 2 (PSK2) is a 1001 amino acid long kinase encoded at 17q11.2 near the neurofibromatosis 1 (NF1) gene (van Woerden et al., 2021). TAOK1 protein is mostly localized in the cytosol and cytoskeleton (Yustein et al., 2003). TAOKs are members of the sterile 20 (STE20) Serine/Threonine kinase family and are comprised of TAOK1, TAOK2, and TAOK3 (Fang et al., 2020). All three TAOKs have similar domain structures with a conserved domain of 250 amino acids long kinase domain at the N-terminus, and a proposed central domain, and C-terminal regulatory domain (Hu et al., 2021). The coiled-coil regions C-terminal to the kinase domain in TAOK1 are also conserved among human, rat, worms, flies, and fish (Beeman et al., 2023). Currently, S181 within the kinase domain is considered an autophosphorylation site of TAOK1 (Byeon et al., 2022). Moreover, the phosphosites outside the kinase domain such as T502, T643, T785, and S990, have also been linked to TAOK1 enzyme activity (Raman et al., 2007). Two TAOK1 isoforms have been identified (alpha and beta) (Hu et al., 2021) and conserved Ser/Thr residues followed by Gln (pSQ/pTQ) in their C-terminal region are considerably phosphorylation sites for ATM (Ataxia Telangiectasia Mutated), and ATR (ATM- and Rad3-Related) kinases ((Moore et al., 2000; Zihni et al., 2006). Phosphorylation and activation of TAOK1 by ATM in response to ultraviolet (UV), infra-red (IR) radiations, and hydroxyurea are associated with DNA damage response and are proposed as an intermediate that link specific G-protein-coupled receptors (GPCRs) to the p38 mitogen-activated protein kinase (MAPK) pathway (Giacomini et al., 2018). Mutation or knockdown of TAOK1 results in inhibition of p38 activation, DNA damage-induced Growth 2 phase (G2)/Mitosis (M) checkpoint, and increased sensitivity to IR radiations (Raman et al., 2007). In response to actin signals, TAOK1 can phosphorylate microtubule-associated proteins involved in regulating interphase microtubule stability and maintain the microtubule dynamics in mitosis to ensure accurate chromosome segregation, and chromosome–microtubule attachment. Supporting this, TAOK1 depletion reduced the density of growing microtubules during interphase and mitosis (Johne et al., 2008, Shrestha et al., 2014). As a core member of the hippo signaling pathway, TAOK1 is linked to the phosphorylation and activation of Mammalian STE20-like protein kinase 2 (MST2) in mammals. Regulation of the Salvador-Warts-Hippo (SWH) pathway (Poon et al., 2011 (Boggiano et al., 2011) is also linked to the regulation of MAPKs (Giacomini et al., 2018). The SWH pathway is often linked to human cancers and for their regulatory role, TAOKs are considered potential tumor suppressors (Plouffe et al., 2016, Poon et al., 2011).

With a significant role in early brain development, differentiation of neurons, and formation of neuronal polarity, dysregulation of TAOK1 is associated with neurological disorders (Dulovic-Mahlow et al., 2019; Wang, J. et al., 2023; Yu et al., 2022). Chromosome 17 microdeletion, consisting of TAOK1, has been linked to retardation in development and microcephaly (Xie et al., 2016). Further, several sequence variations in TAOK1 have been associated with neurological developmental disorders (Beeman et al., 2023, Dulovic-Mahlow et al., 2019, Hunter et al., 2022, Satterstrom et al., 2020, van Woerden et al., 2021, Yu et al., 2022). Currently, a kinase activity-associated phosphorylation in TAOK1 (S181) is also linked to tau pathology in Alzheimer's and frontotemporal lobar degeneration (Giacomini et al., 2018). A novel small molecule TAOK Inhibitor has been shown to decrease tau phosphorylation at T123 and T427 identified in Alzheimer's and also other pathological sites (S262/S356 and S202/T205/S208) in cell line models (Koo et al., 2017). The Mammalian STE20-like protein kinase 3 (MST3) is reported to phosphorylate TAOK1 at T440 and promote phosphorylation-dependent interaction with Myosin Va for its recruitment in dendrites. TAOK1/2 in a protein complex with Myosin Va, Myosin light chain 6, and Drebrin E are associated with cytoskeletal dynamics in neuronal morphogenesis (Ultanir et al., 2014). Recently, Beeman et al., 2023, have identified that mutations that render TAOK1 catalytically inactive induce the plasma membrane phosphoinositide association of TAOK1 to promote membrane protrusions. TAOK1 kinase activity represented autophosphorylation of T440 and T443 residues have been suggested as a molecular switch in this process (Beeman et al., 2023).

Despite its diverse role as a cytoplasmic or membrane-associated kinase and with pivotal role in the regulation of nuclear proteins, TAOK1 remains relatively understudied, with limited information on its substrates, upstream kinases, and interacting partners. This lack of information on TAOK1 extends to the regulatory network of its phosphosites, highlighting a gap in our understanding of the molecular intricacies and regulatory networks involving TAOK1. In this study, we aimed to comprehensively analyze phosphoproteome datasets with differential regulation of TAOK1 phosphosites to select the predominant phosphosites detected and differentially regulated in most biological conditions to infer their role in TAOK1 functions. Towards this, we derived significant positively and negatively correlated phosphosites in other proteins including their known and predicted interactors, substrates, and upstream kinases based on a systematic and robustly extendable approach.