Abstract
Elevated expression of human enhancer filamentation 1 (HEF1; also known as NEDD9 or Cas-L) is an essential stimulus for the metastatic process of various solid tumors. This process requires HEF1 localization to focal adhesions (FAs). Although the association of HEF1 with FAs is considered to play a role in cancer cell migration, the mechanism targeting HEF1 to FAs remains unclear. Moreover, up-regulation of Polo-like kinase 1 (Plk1) positively correlates with human cancer metastasis, yet how Plk1 deregulation promotes metastasis remains elusive. Here, we report that casein kinase 1δ (CK1δ) phosphorylates HEF1 at Ser-780 and Thr-804 and that these phosphorylation events promote a physical interaction between Plk1 and HEF1. We found that this interaction is critical for HEF1 translocation to FAs and for inducing migration of HeLa cells. Plk1-docking phosphoepitopes were mapped/confirmed in HEF1 by various methods, including X-ray crystallography, and mutated for functional analysis in HeLa cells. In summary, our results reveal the role of a phosphorylation-dependent HEF1–Plk1 complex in HEF1 translocation to FAs to induce cell migration. Our findings provide critical mechanistic insights into the HEF1–Plk1 complex–dependent localization of HEF1 to FAs underlying the metastatic process and may therefore contribute to the development of new cancer therapies.
Highlights
Elevated expression of human enhancer filamentation 1 (HEF1; known as NEDD9 or Cas-L) is an essential stimulus for the metastatic process of various solid tumors
We demonstrate that HEF1 phosphorylation at Ser-780 and Thr-804 residues by Casein kinase 1 (CK1)␦ induces the HEF1–Polo-like kinase 1 (Plk1) complex formation
As events in cell migration occur mostly before mitosis, and as HEF1 forms a complex with Plk1 throughout the cell cycle (Fig. 1, A and B), the HEF1–Plk1 functional complex required for HEF1 travel might be assembled in the early stages of the cell cycle
Summary
Phosphorylation-dependent interaction between the C-terminal region of HEF1 and Plk. In line with the importance of pSer-780 and pThr-804 epitopes to PBD binding, both of these residues were conserved across various species (Fig. 2C), and a mass spectrometry analysis revealed the presence of both pSer-780 and pThr-804 peptides in thymidine-treated HeLa cells (Table 1 and Fig. S1A) Their phosphorylation in vivo was further confirmed in immunoprecipitation (IP)-immunoblotting analyses using phospho-specific antibodies, which were generated against either the pSer-780 or the pThr-804 epitope (Fig. 2D). We observed that the expression of CK1␦ efficiently induced pSer-780 and pThr-804 epitopes in a kinase overexpression experiment, whereas, by comparison with CK1␦, CK1⑀ changed these phosphoepitopes to a lesser degree (Fig. 4A) In line with these observations, clear enhancement of Plk PBD binding to FLAG-HEF1 T6 was observed in the CK1␦-overexpressing sample.
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