Zhao Huzi, Zhang Lina, Zhang Yongchen, Zhao Lei, Wan Qing, Wang Bei, Bu Xiaodong, Wan Meiling, Shen Chuanlu
Department of Pathology and Pathophysiology, Medical School, Southeast University, Nanjing, Jiangsu, People's Republic of China.
Oncotarget. 2017 May 30;8(22):36383-36398. doi: 10.18632/oncotarget.16756.
The hominoid oncoprotein TBC1D3 enhances growth factor (GF) signaling and GF signaling, conversely, induces the ubiquitination and subsequent degradation of TBC1D3. However, little is known regarding the regulation of this degradation, and the role of TBC1D3 in the progression of tumors has also not been defined. In the present study, we demonstrated that calmodulin (CaM), a ubiquitous cellular calcium sensor, specifically interacted with TBC1D3 in a Ca2+-dependent manner and inhibited GF signaling-induced ubiquitination and degradation of the oncoprotein in both cytoplasm and nucleus of human breast cancer cells. The CaM-interacting site of TBC1D3 was mapped to amino acids 157~171, which comprises two 1-14 hydrophobic motifs and one lysine residue (K166). Deletion of these motifs was shown to abolish interaction between TBC1D3 and CaM. Surprisingly, this deletion mutation caused inability of GF signaling to induce the ubiquitination and subsequent degradation of TBC1D3. In agreement with this, we identified lysine residue 166 within the CaM-interacting motifs of TBC1D3 as the actual site for the GF signaling-induced ubiquitination using mutational analysis. Point mutation of this lysine residue exhibited the same effect on TBC1D3 as the deletion mutant, suggesting that CaM inhibits GF signaling-induced degradation of TBC1D3 by occluding its ubiquitination at K166. Notably, we found that TBC1D3 promoted the expression and activation of MMP-9 and the migration of MCF-7 cells. Furthermore, interaction with CaM considerably enhanced such effect of TBC1D3. Taken together, our work reveals a novel model by which CaM promotes cell migration through inhibiting the ubiquitination and degradation of TBC1D3.
类人猿癌蛋白TBC1D3增强生长因子（GF）信号传导，相反，GF信号传导会诱导TBC1D3的泛素化及随后的降解。然而，关于这种降解的调控知之甚少，TBC1D3在肿瘤进展中的作用也尚未明确。在本研究中，我们证明钙调蛋白（CaM），一种普遍存在的细胞钙传感器，以Ca2+依赖的方式与TBC1D3特异性相互作用，并抑制人乳腺癌细胞胞质和细胞核中GF信号诱导的该癌蛋白的泛素化和降解。TBC1D3与CaM相互作用的位点定位于氨基酸157171，其包含两个114疏水基序和一个赖氨酸残基（K166）。这些基序的缺失显示会消除TBC1D3与CaM之间的相互作用。令人惊讶的是，这种缺失突变导致GF信号无法诱导TBC1D3的泛素化及随后的降解。与此一致，我们通过突变分析确定TBC1D3的CaM相互作用基序内的赖氨酸残基166是GF信号诱导泛素化的确切位点。该赖氨酸残基的点突变对TBC1D3的影响与缺失突变体相同，表明CaM通过阻断TBC1D3在K166处的泛素化来抑制GF信号诱导的TBC1D3降解。值得注意的是，我们发现TBC1D3促进MMP-9的表达和激活以及MCF-7细胞的迁移。此外，与CaM的相互作用显著增强了TBC1D3的这种作用。综上所述，我们的工作揭示了一种新的模式，即CaM通过抑制TBC1D3的泛素化和降解来促进细胞迁移。
