The crescent-like Golgi ribbon is shaped by the Ajuba/PRMT5/Aurora-A complex-modified HURP.

BACKGROUND: Golgi apparatus (GA) is assembled as a crescent-like ribbon in mammalian cells under immunofluorescence microscope without knowing the shaping mechanisms. It is estimated that roughly 1/5 of the genes encoding kinases or phosphatases in human genome participate in the assembly of Golgi ribbon, reflecting protein modifications play major roles in building Golgi ribbon. METHODS: To explore how Golgi ribbon is shaped as a crescent-like structure under the guidance of protein modifications, we identified a protein complex containing the scaffold proteins Ajuba, two known GA regulators including the protein kinase Aurora-A and the protein arginine methyltransferase PRMT5, and the common substrate of Aurora-A and PRMT5, HURP. Mutual modifications and activation of PRMT5 and Aurora-A in the complex leads to methylation and in turn phosphorylation of HURP, thereby producing HURP p725. The HURP p725 localizes to GA vicinity and its distribution pattern looks like GA morphology. Correlation study of the HURP p725 statuses and GA structure, site-directed mutagenesis and knockdown-rescue experiments were employed to identify the modified HURP as a key regulator assembling GA as a crescent ribbon. RESULTS: The cells containing no or extended distribution of HURP p725 have dispersed GA membranes or longer GA. Knockdown of HURP fragmentized GA and HURP wild type could, while its phosphorylation deficiency mutant 725A could not, restore crescent Golgi ribbon in HURP depleted cells, collectively indicating a crescent GA-constructing activity of HURP p725. HURP p725 is transported, by GA membrane-associated ARF1, Dynein and its cargo adaptor Golgin-160, to cell center where HURP p725 forms crescent fibers, binds and stabilizes Golgi assembly factors (GAFs) including TRIP11, GRASP65 and GM130, thereby dictating the formation of crescent Golgi ribbon at nuclear periphery. CONCLUSIONS: The Ajuba/PRMT5/Aurora-A complex integrates the signals of protein methylation and phosphorylation to HURP, and the HURP p725 organizes GA by stabilizing and recruiting GAFs to its crescent-like structure, therefore shaping GA as a crescent ribbon. Therefore, the HURP p725 fiber serves a template to construct GA according to its shape. Video Abstract.

The JMJD6/HURP axis promotes cell migration via NF-κB-dependent centrosome repositioning and Cdc42-mediated Golgi repositioning.

Golgi apparatus (GA) and centrosome reposition toward cell leading end during directional cell migration in a coupling way, thereby determining cell polarity by transporting essential factors to the proximal plasma membrane. The study provides mechanistic insights into how GA repositioning (GR) is regulated, and how GR and centrosome repositioning (CR) are coupled. Our previous published works reveals that PRMT5 methylates HURP at R122 and the HURP m122 inhibits GR and cell migration by stabilizing GA-associated acetyl-tubulin and then rigidifying GA. The current study further shows that the demethylase JMJD6-guided demethylation of HURP at R122 promotes GR and cell migration. The HURP methylation mimicking mutant 122 F blocks JMJD6-induced GR and cell migration, suggesting JMJD6 relays GR stimulating signal to HURP. Mechanistic studies reveal that the HURP methylation deficiency mutant 122 K promotes GR through NF-κB-induced CR and subsequently CR-dependent Cdc42 upregulation, where Cdc42 couples CR to GR. Taken together, HURP methylation statuses provide a unique opportunity to understand how GR is regulated, and the GA intrinsic mechanism controlling Golgi rigidity and the GA extrinsic mechanism involving NF-κB-CR-Cdc42 cascade collectively dictate GR.

The PRMT5/HURP axis retards Golgi repositioning by stabilizing acetyl-tubulin and Golgi apparatus during cell migration.

The Golgi apparatus (GA) translocates to the cell leading end during directional migration, thereby determining cell polarity and transporting essential factors to the migration apparatus. The study provides mechanistic insights into how GA repositioning (GR) is regulated. We show that the methyltransferase PRMT5 methylates the microtubule regulator HURP at R122. The HURP methylation mimicking mutant 122F impairs GR and cell migration. Mechanistic studies revealed that HURP 122F or endogenous methylated HURP, that is, HURP m122, interacts with acetyl-tubulin. Overexpression of HURP 122F stabilizes the bundling pattern of acetyl-tubulin by decreasing the sensitivity of the latter to a microtubule disrupting agent nocodazole. HURP 122F also rigidifies GA via desensitizing the organelle to several GA disrupting chemicals. Similarly, the acetyl-tubulin mimicking mutant 40Q or tubulin acetyltransferase αTAT1 can rigidify GA, impair GR, and retard cell migration. Reversal of HURP 122F-induced GA rigidification, by knocking down GA assembly factors such as GRASP65 or GM130, attenuates 122F-triggered GR and cell migration. Remarkably, PRMT5 is found downregulated and the level of HURP m122 is decreased during the early hours of wound healing-based cell migration, collectively implying that the PRMT5-HURP-acetyl-tubulin axis plays the role of brake, preventing GR and cell migration before cells reach empty space.

Enolase 1 differentially contributes to cell transformation in lung cancer but not in esophageal cancer.

Enolase transforms 2-phospho-D-glycerate into phosphoenolpyruvate during glycolysis. The human enolase (ENO) family comprises three members named ENO3, which is restricted to muscle tissues, ENO2, which is neuron- and neuroendocrine tissue-specific, and ENO1, which is expressed in almost all tissues. ENO1 is involved in various types of human cancer, including retinoblastoma, hepatocellular carcinoma, pancreatic cancer, renal cell carcinoma, cholangiocarcinoma and gastric cancer. Furthermore, ENO1 enhances cell transformation in numerous cancer cell lines. It has been reported that ENO1 is involved in various activities that are detrimental to cell transformation, including apoptosis and differentiation. However, a few studies demonstrated that ENO1 can be down- or upregulated in various types of lung cancer, which suggests that ENO1 has an ambiguous role in the development of lung cancer. The present study aimed to investigate the differential influences of ENO1 on various types of cancer, and to clarify the role of ENO1 in lung cancer in particular. Western blotting was performed to assess ENO1 protein expression levels in lung cancer and esophageal cancer tissues. Furthermore, exogenous ENO1 was overexpressed in cell lines derived from various tissues and single cell proliferation, flowcytometric analysis, and western blotting were performed to determine the cell proliferation rate, cell transformation status, cell cycle progression and the expression of cell cycle regulators, such as cyclins and cyclin-dependent kinases, and survival factors, such as MAPK and AKT. The results demonstrated that ENO1 was upregulated in collected panels of lung cancer tissues, but not in esophageal cancer tissues. In addition, overexpression of ectopic ENO1 promoted cell proliferation and survival in lung cancer cell lines, which was not the case in other cells, including an esophageal cell line. Furthermore, mechanistic analyses revealed that ENO1 enhanced cell proliferation by accelerating G1 progression and upregulating G1 phase cyclin-dependent kinase 6 (CDK6), and improved cell survival by upregulating p38 in the MAPK cascade and increasing p-AKT in the AKT cascade, in particular in lung cancer cell lines. Overall, the results from the present study demonstrated that ENO1 may contribute to the development of lung cancers, but not esophageal cancers.

Differential contribution of protein phosphatase 1α to cell transformation of different cell types.

Protein phosphorylation plays roles in cell transformation. Numerous protein kinase enzymes actively participate in the formation of various types of cancer by phosphorylating downstream substrates. Aurora­A is a widely known Serine/Threonine (Ser/Thr) oncogenic kinase, which is upregulated in more than twenty types of human cancer. This enzyme phosphorylates a wide range of substrates. For example, Aurora­A induces cell transformation by phosphorylating hepatoma upregulated protein (HURP) at four serine residues, which in turn decreases the phosphorylated levels of cell­growth suppressive Jun N­terminal kinase (p­JNK). Various protein phosphatase enzymes are considered tumor suppressors by the dephosphorylation and consequent inactivation of their oncogenic substrates. Protein phosphatase 1α (PP1α), for instance, acts on Aurora­A by dephosphorylating its substrates. However, the role of PP1α in cancer progression remains ambiguous. PP1α is overexpressed in several cancer tissues, and induces cell apoptosis and differentiation or it inhibits tumor formation in other types of cells. In addition, positive and negative correlations between PP1α expression and lung cancer development have been documented. These observations suggest the differential regulation of PP1α in various cancer tissues, or propose an ambiguous contribution of PP1α to lung cancer development. In order to investigate these contradictory conclusions, it was reported that the chromosomal region covering the PP1α locus was subjected to DNA alterations, such as gain or loss in various human cancer types by a study based on literature search. Upregulation of PP1α was noted in a collection of lung cancer tissues, and was required for the cell transformation of the lung cancer cell line A549. In contrast to this finding, overexpression of ectopic PP1α inhibited cell proliferation in 293T cells. Mechanistic studies revealed that PP1α activated AKT in A549 cells, whereas it further inactivated AKT and disrupted the HURP/JNK signaling cascade in 293T cells. Collectively, the data indicated that PP1α exerted an oncogenic function in lung cancer, while exhibiting various effects on cell transformation in different types of cells via distinct or opposite mechanisms.

Overexpression of Aurora-A bypasses cytokinesis through phosphorylation of suppressed in lung cancer.

Mitosis is a complicated process by which eukaryotic cells segregate duplicated genomes into two daughter cells. To achieve the goal, numerous regulators have been revealed to control mitosis. The oncogenic Aurora-A is a versatile kinase responsible for the regulation of mitosis including chromosome condensation, spindle assembly, and centrosome maturation through phosphorylating a range of substrates. However, overexpression of Aurora-A bypasses cytokinesis, thereby generating multiple nuclei by unknown the mechanisms. To explore the underlying mechanisms, we found that SLAN, a potential tumor suppressor, served as a substrate of Aurora-A and knockdown of SLAN induced immature cytokinesis. Aurora-A phosphorylates SLAN at T573 under the help of the scaffold protein 14-3-3η. The SLAN phosphorylation-mimicking mutants T573D or T573E, in contrast to the phosphorylation-deficiency mutant T573A, induced higher level of multinucleated cells, and the endogenous SLAN p573 resided at spindle midzone and midbody with the help of the microtubule motor MKLP1. The Aurora-A- or SLAN-induced multiple nuclei was prevented by the knockdown of 14-3-3η or Aurora-A respectively, thereby revealing a 14-3-3η/Aurora-A/SLAN cascade negatively controlling cytokinesis. Intriguingly, SLAN T573D or T573E inactivated and T573A activated the key cytokinesis regulator RhoA. RhoA interacted with SLAN np573, i.e., the nonphosphorylated form of SLAN at T573, which localized to the spindle midzone dictated by RhoA and ECT2. Therefore, we report here that SLAN mediates the Aurora-A-triggered cytokinesis bypass and SLAN plays dual roles in that process depending on its phosphorylation status.