Protein arginine methyltransferase 5 is a potential oncoprotein that upregulates G1 cyclins/cyclin-dependent kinases and the phosphoinositide 3-kinase/AKT signaling cascade.

Increasing evidence suggests that PRMT5, a protein arginine methyltransferase, is involved in tumorigenesis. However, no systematic research has demonstrated the cell-transforming activity of PRMT5. We investigated the involvement of PRMT5 in tumor formation. First, we showed that PRMT5 was associated with many human cancers, through statistical analysis of microarray data in the NCBI GEO database. Overexpression of ectopic PRMT5 per se or its specific shRNA enhanced or reduced cell growth under conditions of normal or low concentrations of serum, low cell density, and poor cell attachment. A stable clone that expressed exogenous PRMT5 formed tumors in nude mice, which demonstrated that PRMT5 is a potential oncoprotein. PRMT5 accelerated cell cycle progression through G1 phase and modulated regulators of G1; for example, it upregulated cyclin-dependent kinase (CDK) 4, CDK6, and cyclins D1, D2 and E1, and inactivated retinoblastoma protein (Rb). Moreover, PRMT5 activated phosphoinositide 3-kinase (PI3K)/AKT and suppressed c-Jun N-terminal kinase (JNK)/c-Jun signaling cascades. However, only inhibition of PI3K activity, and not overexpression of JNK, blocked PRMT5-induced cell proliferation. Further analysis of PRMT5 expression in 64 samples of human lung cancer tissues by microarray and western blot analysis revealed a tight association of PRMT5 with lung cancer. Knockdown of PRMT5 retarded cell growth of lung cancer cell lines A549 and H1299. In conclusion, to the best of our knowledge, we have characterized the cell-transforming activity of PRMT5 and delineated its underlying mechanisms for the first time.

Characterization of cocaine-elicited cell vacuolation: the involvement of calcium/calmodulin in organelle deregulation.

The sizes of organelles are tightly regulated in the cells. However, little is known on how cells maintain the homeostasis of these intracellular compartments. Using cocaine as a model compound, we have characterized the mechanism of deregulated vacuolation in cultured rat liver epithelial Clone 9 cells. The vacuoles were observed as early as 10 min following cocaine treatment. Removal of cocaine led to vacuole degeneration, indicating vacuolation is a reversible process. The vacuoles could devour intracellular materials and the vacuoles originated from late endosome/lysosome as indicated by immunofluorescence studies. Instant calcium influx and calmodulin were required for the initiation of vacuole formation. The unique properties of these late endosome/lysosome-derived vacuoles were further discussed. In summary, cocaine elicited a new type of deregulated vacuole and the involvement of calcium/calmodulin in vacuolation could shed light on prevention or treatment of cocaine-induced cytotoxicity.

Identification of V23RalA-Ser194 as a critical mediator for Aurora-A-induced cellular motility and transformation by small pool expression screening.

Human Aurora kinases have three gene family members: Aurora-A, Aurora-B, and Aurora-C. It is not yet established what the specificity of these kinases are and what signals relayed by their reactions. Therefore, we employed small pool expression screening to search for downstream substrates of Aurora-A. Interestingly, all of the identified Aurora-A substrates were resistant to serve as substrates for Aurora-B or Aurora-C, suggesting that these Aurora family members may have distinct substrate specificity for propagation of diverse signaling pathways, even though they share a conserved catalytic kinase domain. Of the candidate substrates, Aurora-A could increase the functional activity of RalA. Mutational analysis revealed that RalA-Ser194 was the phosphorylation site for Aurora-A. Ectopic expression of V23RalA-WT could enhance collagen I-induced cell migration and anchorage-independent growth in Madin-Darby canine kidney (MDCK) Aurora-A stable cell lines. In contrast, overexpression of V23RalA-S194A in MDCK Aurora-A-stable cell lines abolished the intrinsic migration and transformation abilities of Aurora-A. To our knowledge, this is the first systematic search for the downstream substrates of Aurora-A kinase. Moreover, these results support the notion that Aurora-A may act in concert with V23RalA through protein phosphorylation on Ser194 to promote collagen I-induced cell motility and anchorage-independent growth in MDCK epithelial cells.

Fbx7 functions in the SCF complex regulating Cdk1-cyclin B-phosphorylated hepatoma up-regulated protein (HURP) proteolysis by a proline-rich region.

F-box proteins, components of SCF ubiquitin-ligase complexes, are believed to be responsible for substrate recognition and recruitment in SCF-mediated proteolysis. F-box proteins that have been identified to function in the SCF complexes to date mostly have substrate-binding motifs, such as WD repeats or leucine-rich repeats in their C termini. However, many F-box proteins lack recognizable substrate-binding modules; whether they can function in the SCF complexes remains unclear. We show here that Fbx7, an F-box protein without WD repeats and leucine-rich repeats, is required for the proteasome-mediated proteolysis of the hepatoma up-regulated protein (HURP). Depletion of Fbx7 by small interfering RNA leads to depression of HURP ubiquitination and accumulation of HURP abundance. In the SCF(Fbx7) complex, Fbx7 recruits HURP through its C-terminal proline-rich region in a Cdk1-cyclin B-phosphorylation dependent manner. Mutation of the multiple Cdk1-cyclin B phosphorylation sites on HURP or the proline-rich region of Fbx7 abolishes the association between Fbx7 and HURP. Thus, Fbx7 is a functional adaptor of the SCF complex with a proline-rich region as the substrate-binding module. In addition to Fbx7, data base analyses reveal two putative mammalian proline-rich region-containing F-box proteins, KIAA1783 and RIKEN cDNA 2410015K21. Taken together, these findings further expound the diverse substrate-recognition abilities of the SCF complexes.