sAP4, a Cytoplasmic Isoform of Transcription Factor AP4, Functions as a Novel Nucleation Promoting Factor in Enhancing Actin Polymerization for Cell Migration

轉錄因子AP4的一個細胞質亞型,sAP4是在細胞移動中作用於增強肌動蛋白聚合的新型細胞成核促進因子

Student thesis: Doctoral Thesis

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Award date4 Sep 2019

Abstract

AP4 (transcription factor activating enhancer-binding protein 4) is a transcriptional factor involved in regulating many cellular functions such as cell growth, proliferation, EMT and apoptosis. As established in many studies, AP4 predominantly localizes in the nucleus. Recently, several isoforms of AP4 have been reported, and one of them termed the short form-AP4 (sAP4) is the focus of this study. The amino acid sequence of sAP4 is identical to full-length AP4 without the N-terminal region up to the DNA-binding domain. sAP4 was first identified from the cDNA of a neuroblastoma sample from a patient. However, the potential functions of sAP4 are totally unknown. Surprisingly, here we report that sAP4, a cytoplasmic form of AP4, promoted membrane ruffling and cell migration, a process in the leading edge protrusion via the elongation of actin filaments. This work was initiated by looking for interacting partners of sAP4 that may be involved in events associated with actin filament dynamics.

To locate sAP4 in the actin-based protrusive structures and to observe whether sAP4 co-localizes with any known regulatory proteins involved in mediating actin polymerization. I constructed a series of deletion mutants of sAP4 in a GST-tagged vector for bacterial expression and a FLAG-tagged vector for mammalian expression. In addition, I constructed various identified actin binding and regulatory proteins in both His- and GFP-tagged versions for bacterial and mammalian expression, respectively. These proteins include cortactin (WT and SH3-deletion mutant), profilin (WT and H133S mutant), and ARP3. Subsequently, I did transient co-transfection of FLAG-tag-sAP4 (wild-type and all serial deletion mutants) expression vector and expression vector containing the genes of cortactin (V5-tag), profilin (VSV-tag), and ARP3 (VSV-tag) (3 major actin interacting proteins) into mammalian cells. Then I used immunofluorescence microscopy to determine the colocalization of sAP4 and its interacting partners in the cell front. As expected, sAP4 was found in the leading edge of the cells and colocalizes with cortactin, profilin, and ARP3.

By the use of both direct binding (or pulldown) assay and immunoprecipitation method, we found that the proline-rich domain (PRD) in sAP4 is important for its interaction with cortactin and profilin. In addition, we mapped the proline-rich motif in sAP4 to be critical for binding to the src homology 3 (SH3) domain in cortactin and a specific polyproline-binding site on profilin. These results support the hypothesis that sAP4 provides a link between the cytoskeleton assembly and the signal transduction pathway in migrating cells.

We found that sAP4 binds tightly with the ARP3 in both the lysate of the transiently transfected cells and in their purified recombinant form. Further, I discovered that sAP4 bound to the ARP3 complex via the acidic amino acid-rich region (AC1) in sAP4, which is similar in amino acid sequence to those found in all actin nucleation-promoting factors (NPFs), such as cortactin, ActA, Abp1p, SCAR, WAVE, N-WASP and WASP proteins. The deletion of the conserved acidic residues of sAP4 abolished its interaction with the ARP2/3 complex, suggesting sAP4 is a novel interacting partner for the activation of the ARP2/3 complex.

Based on the results of the previous section, we tested the direct effect of sAP4 on the activity of actin polymerization in vitro using the pyrene-labelled actin polymerization assay. In vitro, sAP4 alone had no obvious effect on the enhancement of actin polymerization. sAP4 in combination with the ARP2/3 complex significantly increased the rate of actin polymerization suggesting that sAP4 functions similar to other nucleation promoting factors proteins such as WASP and cortactin in activating of the ARP2/3 complex to promote actin polymerization.

In conclusion, we propose a model on how sAP4 interacts with actin regulatory proteins to promote ruffle formation and cell migration. The stimulation of growth factor receptor on the plasma membrane by its ligand promotes the formation of the GTP-bound form of RAC and RHOD, which may result in the interaction with sAP4. sAP4 colocalizes to the cell periphery where it uses the proline-rich motif to bind the SH3 domain of cortactin, which in turn stabilizes the actin filament assembly and activates the ARP2/3 complex. The direct binding of sAP4 to profilin results in the recruitment of monomeric actin to the barb ends of growing actin filaments. sAP4 enhances the actin branch formation by its direct binding and activation of the ARP2/3 complex. Our results indicate a novel role of sAP4 as a nucleation promoting factor in enhancing actin polymerization, which in turn affects cell migration. This study suggests that sAP4 may act as a potential therapeutic target to disrupt actin cytoskeleton and prevent the spreading of migrating cancer cells into other tissues.

    Research areas

  • Transcription factors, TFAP4, Cell migration