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

## TL;DR

This study explores how TGFβ signaling influences autophagy during palate development by analyzing phosphorylation events in mouse cells.

## Contribution

The study identifies novel TGFβ-dependent phosphorylation events and a new role for CSNK2A in regulating phospho-signaling during palatogenesis.

## Key findings

- TGFβ2 treatment altered the MEPM phosphoproteome, with 477 upregulated and 53 downregulated phosphopeptides.
- Seven serine and one threonine phosphorylation motifs were enriched in TGFβ2-treated cells.
- CSNK2A, MAPKs, and CDKs were identified as key regulators of TGFβ-dependent phosphorylation.

## Abstract

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.