Abstract
Twist1 is a basic helix-loop-helix transcription factor that strongly promotes epithelial-to-mesenchymal transition, migration, invasion, and metastasis of cancer cells. The MAPK-phosphorylated Twist1 on its serine 68 (Ser(P)(68)-Twist1) has a significantly enhanced stability and function to drive cancer cell invasion and metastasis. However, the phosphatase that dephosphorylates Ser(P)(68)-Twist1 and destabilizes Twist1 has not been identified and characterized. In this study, we screened a serine/threonine phosphatase cDNA expression library in HEK293T cells with ectopically coexpressed Twist1. We found that the small C-terminal domain phosphatase 1 (SCP1) specifically dephosphorylates Ser(P)(68)-Twist1 in both cell-free reactions and living cells. SCP1 uses its amino acid residues 43-63 to interact with the N terminus of Twist1. Increased SCP1 expression in cells decreased Ser(P)(68)-Twist1 and total Twist1 proteins, whereas knockdown of SCP1 increased Ser(P)(68)-Twist1 and total Twist1 proteins. Furthermore, the levels of SCP1 are negatively correlated with Twist1 protein levels in several cancer cell lines. SCP1-dephosphorylated Twist1 undergoes fast degradation via the ubiquitin-proteasome pathway. Importantly, an increase in SCP1 expression in breast cancer cells with either endogenous or ectopically expressed Twist1 largely inhibits the Twist1-induced epithelial-to-mesenchymal transition phenotype and the migration and invasion capabilities of these cells. These results indicate that SCP1 is the phosphatase that counterregulates the MAPK-mediated phosphorylation of Ser(68)-Twist1. Thus, an increase in SCP1 expression and activity may be a useful strategy for eliminating the detrimental roles of Twist1 in cancer cells.
Highlights
Nowadays more than 90% of cancer deaths are attributed to metastasis acquired through multiple steps, including cancer cell local migration and invasion, intravasation into and extravasation out of the circulating systems, and colonization in a distant organ site [1,2,3]
We found that small C-terminal domain phosphatase 1 (SCP1) expression significantly decreased the level of Ser(P)68-Twist1-F as well as the level of total Twist1-F protein, whereas other phosphatases showed no obvious effect on the level of Ser(P)68-Twist1-F (Fig. 1A and data not shown)
Western blotting analysis revealed that both Ser(P)68-Twist1-F and total Twist1-F were decreased in a FLAG-tagged SCP1 (F-SCP1) dose-dependent manner when total protein inputs of all samples were normalized to GAPDH (Fig. 1B)
Summary
Plasmids—Human SCP1 and its dominant negative mutant (dnSCP1) cDNAs in the pGEX-4T-1 plasmid were described previously [24]. For the in vivo phosphatase assay, cells were transfected with SCP1 or mutant SCP1 expression plasmids using Lipofectamine 2000 reagent (Life Technologies). Ubiquitination Assay—HEK293 cell lines (105 cells) with DOX-inducible Twist1-FLAG expression were transfected with 1 g of HA-tagged ubiquitin (HA-ubiquitin) plasmid and HA-SCP1 or HA-dnSCP1 plasmid. ShRNA-based Knockdown of SCP1—MDA-MB-436 and Ishikawa cells were infected with lentiviral particles of the GIPZ lentiviral shRNAmir-GFP system as described previously [19] These lentiviral vectors mediate the expression of non-targeting control shRNA or shRNAs (clone RHS4430-101068525 and clone RHS4430-101071006, Sigma) that target human SCP1 mRNA. The protein levels of SCP1, Twist, and Ser(P)68-Twist in these cells were assayed by Western blotting. Forty-eight hours later, transfected cells were subjected to the growth, migration, and invasion assays using the RT-CES system (ACEA Biosciences) as described previously [12].
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