shCTR vs shTIMAP
The protein phosphatase 1 regulatory subunit TGF-ß inhibited membrane-associated protein (TIMAP) is highly expressed in neuronal cells, yet its role in regulating protein phosphatase 1 (PP1) activity remains poorly understood. To investigate this, TIMAP was stably depleted in SH-SY5Y human neuroblastoma cells using short hairpin RNA. Transcriptomic analysis identified over 600 differentially expressed genes upon TIMAP depletion, with gene ontology analysis highlighting significant disruption of the bone morphogenetic protein (BMP) signaling pathway. Functional studies showed that TIMAP depletion markedly reduced SMAD1 protein levels following stimulation with BMP7, an important ligand within the signaling pathway. Although the C-terminal phosphorylation of SMAD1/5/8 which is required for activation, remained largely unchanged, the nuclear translocation of these activated SMADs was strongly impaired. This reduction in nuclear accumulation reflects the role of TIMAP in directing protein phosphatase 1 to the SMAD1 linker region, thereby controlling the phosphorylation dynamics at a site that recruits the ubiquitin ligase SMURF1. The absence of TIMAP leads to increased SMAD1 ubiquitination and elevated Ser206 phosphorylation in the SMAD1 linker region, resulting in reduced protein levels and impaired nuclear signaling despite normal activation. These findings reveal that TIMAP is essential for maintaining SMAD1 stability by targeting the PP1 catalytic subunit to the SMAD1 linker region to dephosphorylate regulatory sites, thereby preventing ubiquitin-mediated degradation and ensuring proper nuclear function.