Integrated Phosphoproteomics Identifies TGFβ-Dependent Phosphorylation Events Linking Kinase Signaling to Autophagy in Palatogenesis

Background: Cleft palate (CP) is a prevalent craniofacial malformation, with the TGFβ pathway playing a critical role. Recent evidence links autophagy to regulating mouse embryonic palatal mesenchyme (MEPM) cells, but its interaction with TGFβ-activated phosphorylation cascades remains largely unknown. Here, we investigated the interplay between these pathways during palatogenesis. Methods: H&E and IHC analyses revealed increased expression of Beclin 1 and LC3 during the critical period of palatal shelf elevation and fusion (E13.5–E15.5). Bulk RNA sequencing (Bulk RNA-seq) further revealed enrichment of autophagy-related pathways and their interaction with TGFβ signaling. TMT-based phosphoproteomics was performed on TGFβ2-treated MEPM cells. Results: We identified 23,471 peptides and 3952 proteins, including 6339 phosphopeptides corresponding to 2195 phosphoproteins. Differential analysis found 477 phosphopeptides with increased abundance and 53 with decreased abundance, revealing the enrichment of seven serine (p-Ser) motifs (RxxS, SDxD, SDxE, SP, SxDE, SxEE, SxxxxD) and one threonine (p-Thr) motif (TP). Notably, kinase-substrate enrichment analysis identified CSNK2A as a previously unrecognized phosphorylation regulator, together with MAPKs and CDKs. Functional enrichment showed significant involvement of mTOR, MAPK, and autophagy/mitophagy pathways. Conclusions: Our findings revealed that TGFβ2 reshapes the MEPM phosphoproteome through Smad-independent pathway, expanding the palate-specific phospho-signaling atlas beyond the canonical Smad cascade.