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 GSK3bβ 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.

中文翻译：

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PKA 和 GSK3β 对人 CEACAM1-LF 的磷酸化促进其与 β-连环蛋白的相互作用。

CEACAM1-LF 是一种同型细胞粘附分子，通过 72 个氨基酸的细胞质结构域转导细胞内信号，该结构域包含两个免疫受体酪氨酸抑制基序 (ITIM) 和一个 β-连环蛋白结合位点。在啮齿动物 CEACAM1 中，PKC 对 Ser503 的磷酸化显示通过 ITIM 磷酸化水平影响胆汁酸转运或肝脂肪变性，但人类 CEACAM1 (Ser508) 中等效残基的磷酸化尚不清楚。在这里，我们通过 15N 标记的细胞质域肽的 NMR 分析研究了这种类似的磷酸化。与多种 Ser/Thr 激酶一起孵育显示，GSK3bβ 会磷酸化 Ser508，但 PKC 不会。PKC 缺乏磷酸化可能是由于啮齿动物和人类基因之间的进化序列变化。通过质谱法和 NMR 进行的磷酸化位点分配揭示了 PKA 对 Ser472、Ser461 和 Ser512 的磷酸化，其中 Ser512 是 GSK3β 结合的保守共有位点的一部分。我们在这里表明，只有在 PKA 磷酸化 Ser512 后，GSK3β 才能磷酸化 Ser508。PKA 对 Ser512 的磷酸化促进了与跨越 CEACAM1 的 ITIM 的扩展区域的 β-连环蛋白的犰狳重复结构域的紧密关联。Src 对 ITIM 的磷酸化动力学以及 SHP2 的去磷酸化受 Ser508/512 磷酸化的影响，表明 PKA 和 GSK3β 可能调节人 CEACAM1-LF 的信号转导活性。CEACAM1-LF 与 PKA 促进的 β-连环蛋白的相互作用表明 CEACAM1-LF 的两个 ITIM 之间存在紧密关联。