A Regulatory Element in the Intrinsically Disordered C-Terminal Region of LMTK3 Modulates Its Kinase Domain...

Lemur tail kinase 3 (LMTK3) is an oncogenic Ser/Thr kinase implicated in breast cancer (BC) progression, therapy resistance, and poor clinical outcomes, yet the molecular mechanisms governing its regulation remain poorly understood, particularly the role of its C-terminal intrinsically disordered region (IDR). Given that IDRs frequently harbour hidden structural motifs that control protein dynamics, we combined computational, biophysical, and biochemical approaches to systematically map regulatory elements within the LMTK3 C-terminus, identifying two regions (residues 688-1095 and 1181-1486) that interact with the LMTK3 kinase domain (LMTK3-KD). Characterisation of these interactions revealed that LMTK3^1181-1486 displays preferential binding to inactive wild-type LMTK3-KD over a constitutively active mutant (LMTK3-KD^L313R), a behaviour consistent with a potential autoinhibitory interaction. Guided by AlphaFold3 modelling, we localised this interaction primarily to a short α-helical motif (α-helix 2; residues 1247-1258) within the C-terminal IDR and subsequently identified Ser1258 within this motif as a candidate regulatory phosphorylation site, using [γ-³²P]-ATP kinase assays and mass spectrometry. Phosphorylation at Ser1258 altered interactions between α-helix 2 and the kinase domain, reducing binding to wild-type LMTK3-KD while increasing affinity for LMTK3-KD^L313R. Functionally, phospho-null mutation of Ser1258 impaired oestrogen receptor alpha (ERα) upregulation, proliferation, migration, and clonogenicity in ER-positive BC cell lines, and reduced tumour growth in female BALB/c nude mice bearing orthotopic MCF7 xenografts. Together, these findings identify a previously uncharacterised regulatory element within the LMTK3 C-terminus and support a model in which Ser1258 phosphorylation modulates kinase domain interactions and LMTK3-driven oncogenic functions in BC.