IRBIT is a multifunctional protein that controls the activity of various epithelial ion transporters including NBCe1-B. Interaction with IRBIT increases NBCe1-B activity and exposes two cryptic Cl⁻-sensing GXXXP sites that enable regulation of NBCe1-B by intracellular Cl⁻ (Cl⁻_in). Here, phosphoproteomic analysis revealed that IRBIT controlled five phosphorylation sites in NBCe1-B that determined both the active conformation of the transporter and its regulation by Cl⁻_in. Mutational analysis suggested that the phosphorylation status of Ser²³², Ser²³³, and Ser²³⁵ was regulated by IRBIT and determined whether NBCe1 transporters are in active or inactive conformations. The absence of phosphorylation at Ser²³², Ser²³³, or Ser²³⁵ produced NBCe1-B in the conformations pSer²³³/pSer²³⁵, pSer²³²/pSer²³⁵, or pSer²³²/pSer²³³, respectively. The activity of the pSer²³³/pSer²³⁵ form was similar to that of IRBIT-activated NBCe1-B, but it was insensitive to inhibition by Cl⁻_in. The properties of the pSer²³²/pSer²³⁵ form were similar to those of wild-type NBCe1-B, whereas the pSer²³²/pSer²³³ form was partially active, further activated by IRBIT, but retained inhibition by Cl⁻_in. Furthermore, IRBIT recruited the phosphatase PP1 and the kinase SPAK to control phosphorylation of Ser⁶⁵, which affected Cl⁻_in sensing by the ³²GXXXP³⁶ motif. IRBIT also recruited the phosphatase calcineurin and the kinase CaMKII to control phosphorylation of Ser¹², which affected Cl⁻_in sensing by the ¹⁹⁴GXXXP¹⁹⁸ motif. Ser²³², Ser²³³, and Ser²³⁵ are conserved in all NBCe1 variants and affect their activity. These findings reveal how multiple kinase and phosphatase pathways use phosphorylation sites to fine-tune a transporter, which have important implications for epithelial fluid and HCO₃⁻ secretion.

IRBIT 是一种多功能蛋白，可控制包括 NBCe1-B 在内的各种上皮离子转运蛋白的活性。与 IRBIT 的相互作用增加了 NBCe1-B 活性并暴露了两个神秘的 Cl ^-感应 GXXXP 位点，这些位点能够通过细胞内 Cl ^- (Cl ^- _in )调节 NBCe1-B 。在这里，磷酸化蛋白质组学分析显示 IRBIT 控制着 NBCe1-B 中的五个磷酸化位点，这决定了转运蛋白的活性构象及其受 Cl ^- _{in 的}调节。突变分析表明Ser ²³²、Ser ²³³和Ser ²³⁵的磷酸化状态受 IRBIT 调节并确定 NBCe1 转运蛋白是处于活性还是非活性构象。Ser ²³²、Ser ²³³或Ser ²³⁵没有磷酸化产生分别为pSer ²³³ /pSer ²³⁵、pSer ²³² /pSer ²³⁵或pSer ²³² /pSer ²³³构象的NBCe1-B 。pSer ²³³ /pSer ²³⁵形式的活性与 IRBIT激活的 NBCe1-B 相似，但对 Cl ^- _{in 的}抑制不敏感。pSer ²³² /pSer ^{235 的特性}形式与野生型 NBCe1-B 相似，而 pSer ²³² /pSer ²³³形式具有部分活性，被 IRBIT 进一步激活，但保留了 Cl ^- _{in 的}抑制作用。此外，IRBIT 募集磷酸酶 PP1 和激酶 SPAK 来控制 Ser ^{65 的}磷酸化，这会影响 Cl ^-_在³² GXXXP ³⁶基序的感应中。IRBIT 还招募了磷酸酶钙调神经磷酸酶和激酶 CaMKII 来控制 Ser ^{12 的}磷酸化，这会影响 Cl ^-通过¹⁹⁴ GXXXP ¹⁹⁸基序_进行感应。丝氨酸²³²、Ser ²³³和Ser ²³⁵在所有 NBCe1 变体中都是保守的并影响它们的活性。这些发现揭示了多种激酶和磷酸酶途径如何使用磷酸化位点来微调转运蛋白，这对上皮液和 HCO ₃ ^-分泌具有重要意义。