Latchford Lauren P, Perez Liz S, Conage-Pough Jason E, Turk Reem, Cusimano Marissa A, Vargas Victoria I, Arora Sonal, Shienvold Sophia R, Kulp Ryan R, Belverio Hailey M, White Forest M, Thévenin Anastasia F
Department of Biological Sciences, Moravian University, Bethlehem, Pennsylvania, USA.
Koch Institute for Integrative Cancer Research and Department of Biological Engineering, MIT, Cambridge, Massachusetts, USA.
J Biol Chem. 2025 Mar;301(3):108178. doi: 10.1016/j.jbc.2025.108178. Epub 2025 Jan 10.
Phosphorylation of connexin 43 (Cx43) is an important regulatory mechanism of gap junction (GJ) function. Cx43 is modified by several kinases on over 15 sites within its ∼140 amino acid-long C-terminus (CT). Phosphorylation of Cx43CT on S255, S262, S279, and S282 by ERK has been widely documented in several cell lines, by many investigators. Phosphorylation of these sites by JNK and p38, on the other hand, is not well-established. Indeed, ERK is a kinase activated by growth factors and is upregulated in diseases, such as cancer. JNK and p38, however, have a largely tumor-suppressive function due to their stress-activated and apoptotic role. We investigated substrate specificity of all three MAPKs toward Cx43CT, first by using purified proteins, and then in two cell lines (MDCK - non-cancerous, epithelial cells and porcine PAECs-pulmonary artery endothelial cells). Cx43 phosphorylation was monitored through gel-shift assays on an SDS-PAGE, immunodetection with phospho-Cx43 antibodies, and LC-MS/MS phosphoproteomic analyses. Our results demonstrate that p38 and JNK specificity differ from each other and from ERK. JNK has a strong preference for S255, S262, and S279, while p38 readily phosphorylates S262, S279, and S282. While we confirmed that ERK can phosphorylate all four serines (255, 262, 279, and 282), we also identified T290 as a novel ERK phosphorylation site. In addition, we assessed Cx43 GJ function upon activation or inhibition of each MAPK in PAECs. This work underscores the importance of delineating the effects of ERK, JNK, and p38 signaling on Cx43 and GJ function.
连接蛋白43（Cx43）的磷酸化是间隙连接（GJ）功能的重要调节机制。Cx43在其约140个氨基酸长的C末端（CT）内的15个以上位点被多种激酶修饰。ERK对Cx43CT的S255、S262、S279和S282位点的磷酸化已在多种细胞系中被众多研究者广泛报道。另一方面，JNK和p38对这些位点的磷酸化作用尚未完全明确。事实上，ERK是一种由生长因子激活的激酶，在癌症等疾病中上调。然而，JNK和p38由于其应激激活和凋亡作用，在很大程度上具有肿瘤抑制功能。我们首先使用纯化蛋白，然后在两种细胞系（MDCK - 非癌性上皮细胞和猪肺动脉内皮细胞PAECs）中研究了所有三种丝裂原活化蛋白激酶（MAPK）对Cx43CT的底物特异性。通过SDS - PAGE上的凝胶迁移分析、用磷酸化Cx43抗体进行免疫检测以及液相色谱 - 串联质谱（LC - MS/MS）磷酸化蛋白质组分析来监测Cx43的磷酸化。我们的结果表明，p38和JNK的特异性彼此不同，且与ERK也不同。JNK强烈倾向于S255、S262和S279，而p38容易磷酸化S262、S279和S282。虽然我们证实ERK可以磷酸化所有四个丝氨酸（255、262、279和282），但我们也将T290鉴定为一个新的ERK磷酸化位点。此外，我们评估了PAECs中每种MAPK激活或抑制后Cx43的GJ功能。这项工作强调了阐明ERK、JNK和p38信号传导对Cx43和GJ功能影响的重要性。
