Research Centre for Natural Sciences, Institute of Enzymology, 1117 Budapest, Hungary.
Department of Biochemistry, Eötvös Loránd University, 1117 Budapest, Hungary.
Int J Mol Sci. 2021 Jul 28;22(15):8103. doi: 10.3390/ijms22158103.
The scaffold protein Tks4 is a member of the p47-related organizer superfamily. It plays a key role in cell motility by being essential for the formation of podosomes and invadopodia. In addition, Tks4 is involved in the epidermal growth factor (EGF) signaling pathway, in which EGF induces the translocation of Tks4 from the cytoplasm to the plasma membrane. The evolutionarily-related protein p47 and Tks4 share many similarities in their -terminal region: a phosphoinositide-binding PX domain is followed by two SH3 domains (so called "tandem SH3") and a proline-rich region (PRR). In p47, the PRR is followed by a relatively short, disordered -terminal tail region containing multiple phosphorylation sites. These play a key role in the regulation of the protein. In Tks4, the PRR is followed by a third and a fourth SH3 domain connected by a long (~420 residues) unstructured region. In p47, the tandem SH3 domain binds the PRR while the first SH3 domain interacts with the PX domain, thereby preventing its binding to the membrane. Based on the conserved structural features of p47 and Tks4 and the fact that an intramolecular interaction between the third SH3 and the PX domains of Tks4 has already been reported, we hypothesized that Tks4 is similarly regulated by autoinhibition. In this study, we showed, via fluorescence-based titrations, MST, ITC, and SAXS measurements, that the tandem SH3 domain of Tks4 binds the PRR and that the PX domain interacts with the third SH3 domain. We also investigated a phosphomimicking Thr-to-Glu point mutation in the PRR as a possible regulator of intramolecular interactions. Phosphatidylinositol-3-phosphate (PtdIns(3)P) was identified as the main binding partner of the PX domain via lipid-binding assays. In truncated Tks4 fragments, the presence of the tandem SH3, together with the PRR, reduced PtdIns(3)P binding, while the presence of the third SH3 domain led to complete inhibition.
支架蛋白 Tks4 是 p47 相关组织者超家族的成员。它通过形成足突和侵入小体在细胞运动中发挥关键作用。此外，Tks4 参与表皮生长因子 (EGF) 信号通路，EGF 诱导 Tks4 从细胞质易位到质膜。进化相关的蛋白 p47 和 Tks4 在其 C 端区域具有许多相似性：一个磷酸肌醇结合 PX 结构域后面跟着两个 SH3 结构域（所谓的“串联 SH3”）和一个富含脯氨酸的区域（PRR）。在 p47 中，PRR 后面是一个相对较短的、无序的 C 端尾部区域，包含多个磷酸化位点。这些在蛋白质的调节中起着关键作用。在 Tks4 中，PRR 后面是第三个和第四个 SH3 结构域，由一个长（约 420 个残基）的无结构区域连接。在 p47 中，串联 SH3 结构域结合 PRR，而第一个 SH3 结构域与 PX 结构域相互作用，从而阻止其与膜结合。基于 p47 和 Tks4 的保守结构特征以及已经报道的 Tks4 中第三个 SH3 结构域和 PX 结构域之间的分子内相互作用的事实，我们假设 Tks4 受到自身抑制的类似调节。在这项研究中，我们通过荧光滴定、MST、ITC 和 SAXS 测量表明，Tks4 的串联 SH3 结构域结合 PRR，而 PX 结构域与第三个 SH3 结构域相互作用。我们还研究了 PRR 中 Thr 到 Glu 的磷酸模拟点突变作为分子内相互作用的可能调节剂。通过脂质结合测定鉴定 PtdIns(3)P 是 PX 结构域的主要结合伴侣。在截断的 Tks4 片段中，串联 SH3 结构域与 PRR 的存在降低了 PtdIns(3)P 的结合，而第三个 SH3 结构域的存在导致完全抑制。
