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Phosphorylation of human CEACAM1-LF by PKA and GSK3β promote its interaction with β-catenin.

ABSTRACT: CEACAM1-LF, a homotypic cell adhesion adhesion molecule, transduces intracellular signals via a 72 amino acid cytoplasmic domain that contains two immunoreceptor tyrosine based inhibitory motifs (ITIMs) and a binding site for β-catenin. Phosphorylation of Ser503 by PKC in rodent CEACAM1 was shown to affect bile acid transport or hepatosteatosis via the level of ITIM phosphorylation, but the phosphorylation of the equivalent residue in human CEACAM1 (Ser508) was unclear. Here we studied this analogous phosphorylation by NMR analysis of the ¹⁵N labeled cytoplasmic domain peptide. Incubation with a variety of Ser/Thr kinases revealed phosphorylation of Ser508 by PKA but not by PKC. The lack of phosphorylation by PKC is likely due to evolutionary sequence changes between the rodent and human genes. Phosphorylation site assignment by mass spectrometry and NMR revealed phosphorylation of Ser472, Ser461 and Ser512 by PKA, of which Ser512 is part of a conserved consensus site for GSK3β binding. We showed here that only after phosphorylation of Ser512 by PKA was GSK3β able to phosphorylate Ser508. Phosphorylation of Ser512 by PKA promoted a tight association with the armadillo repeat domain of β-catenin at an extended region spanning the ITIMs of CEACAM1. The kinetics of phosphorylation of the ITIMs by Src, as well dephosphorylation by SHP2, were affected by the presence of Ser508/512 phosphorylation, suggesting that PKA and GSK3β may regulate the signal transduction activity of human CEACAM1-LF. The interaction of CEACAM1-LF with β-catenin promoted by PKA is suggestive of a tight association between the two ITIMs of CEACAM1-LF.

SUBMITTER: Hu W

PROVIDER: S-EPMC8564729 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cacumen Platycladi (CP) consists of the dried needles of Platycladus orientalis L.) Franco. It was clinically demonstrated that it effectively regenerates hair, but the underlying mechanism remains unknown. Thus, we employed shaved mice to verify the hair growth-promoting capability of the water extract of Cacumen Platycladi (WECP). The morphological and histological analyses revealed that WECP application could significantly promote hair growth and hair follicles (HFs) construction, in comparison to that of control group. Additionally, the skin thickness and hair bulb diameter were significantly increased by the application of WECP in a dose-dependent manner. Besides, the high dose of WECP also showed an effect similar to that of finasteride. In an in vitro assay, WECP stimulated dermal papilla cells (DPCs) proliferation and migration. Moreover, the upregulation of cyclins (cyclin D1, cyclin-dependent kinase 2 (CDK2), and cyclin-dependent kinase 4 (CDK4)) and downregulation of P21 in WECP-treated cell assays have been evaluated. We identified the ingredients of WECP using ultra-high-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q/TOF-MS) and endeavored to predict their relevant molecular mechanisms by network analysis. We found that the Akt (serine/threonine protein kinase) signaling pathway might be a crucial target of WECP. It has been demonstrated that WECP treatment activated the phosphorylation of Akt and glycogen synthase kinase-3-beta (GSK3β), promoted β-Catenin and Wnt10b accumulation, and upregulated the expression of lymphoid enhancer-binding factor 1 (LEF1), vascular endothelial growth factor (VEGF), and insulin-like growth factor 1 (IGF1). We also found that WECP significantly altered the expression levels of apoptosis-related genes in mouse dorsal skin. The enhancement capability of WECP on DPCs proliferation and migration could be abrogated by the Akt-specific inhibitor MK-2206 2HCl. These results suggested that WECP might promote hair growth by modulating DPCs proliferation and migration through the regulation of the Akt/GSK3β/β-Catenin signaling pathway.

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Phosphorylation of human CEACAM1-LF by PKA and GSK3β promote its interaction with β-catenin.

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