The Nucleolar Protein PAK1IP1 Regulates Cell Cycle Progression And Ribosomal Biogenesis

Cell growth and proliferation are two precisely controlled and integrated processes throughout the whole life of an organism. And the maintenance of an appropriate cell number in a well-functioning organ is dependent on the balance of the rate at which the cells are generated via proliferation and eliminated by apoptosis. Cell proliferation is highly regulated at each phase of the cell cycle, and the G1/S checkpoint is one of the major cell cycle transition points. At the G1/S transition, a cell can decide its fate, such as cell division, G1 arrest, quiescence, or differentiation, in response to diverse signals. Any malfunctions at this checkpoint, such as activation of an oncogene or inactivation of a tumor suppressor gene, may lead to abnormal cell proliferation and transformation, consequently cancer development. One of the most important tumor suppressor genes is TP53, which is mutated in more than 50% of all types of human cancers while the rest of cancers are often associated with alterations of p53 modulators that regulate p53 stability and activity. For example, the mdm2 gene, which encodes a p53 suppressor called MDM2, is upregulated in 7% of human cancers which lack the mutation of p53. In normal cells, p53 level is low mainly due to the regulation by MDM2, which possesses an E3 ubiquitin ligase activity and mediates p53 ubiquitination and degradation through a feedback mechanism. In response to various stresses, such as DNA damage, oncogenic and nucleolar stresses, p53 is stabilized and activated largely through the inhibition of MDM2, leading to cell cycle arrest, apoptosis, DNA repair or senescence.Nucleolar/ribosomal stress is induced by the perturbation of ribosomal biogenesis, such as the interference in rRNA synthesis, processing, and ribosome assembly. It has been reported that the treatment of human cells with fluorouracil (5-FU) or actinomycin D (low doses), serum starvation, disruption of nucleolar or ribosomal proteins leads to ribosomal stress and consequent p53 activation. In response to ribosomal stress, several nucleolar proteins including nucleophosmin, nucleostemin, L5, L11, L23, and S7 can bind to MDM2 and inhibit its activity, leading to p53 stabilization and activation. Hence, these nucleolar proteins play a critical role in transmitting nucleolar stress signals to the p53 pathway, coupling this pathway with ribosomal biogenesis.Our previous study showed that PAK1IP1 is abundantly expressed in most human tissues, and negatively regulates the activity of PAK1 which has been shown to regulate various cellular activities, including cell proliferation, cell survival, mitosis, and transcription. PAK1IP1 contains five G proteinβ-like WD40 repeats and shares high sequence homology with Makll in budding yeast, and Skb15 in fission yeast. The two yeast orthologs of PAK1IP1 are essential for cell viability. Although we demonstrated that mouse Paklipl can substitute Skb15 function in yeast, the biological function of PAK1IP1 in mammalian cells is poorly understood.Based on research reports and hypothesis of PAK1IP1, we performed a series of experiments to study on the mechanism and function of PAK1IP1. The project can be divided into three parts:A. The analysis of cellular localization and expression on PAK1IP1We analyzed the nuclear and nucleolar localization signal sequence(s) (NLS/NoLS) for PAK1IP1 by performing a bioinformatical search. The conserved NLS/NoLS sequences were identified in the C-terminus [from 371 to 389 amino acid residues (KKRKMVEMLEKKRKKKKIK)] of PAK1IP1. We cloned the PAK1IP1 full length by PCR and inserted it into GFP vector. We found PAK1IP1 is a nucleolar protein in mammalian cells by using fluorescence microscopy, and PAK1IP1 proteins exhibited diffused nucleoplasmic distribution with apparently nucleolar accumulation. Deletion mutations of PAK1IP1 GFP fusion constructs were generated to further characterize the function of each NLS motif. The predicted NLS is essential for the nuclear localization of PAK1IP1, and the three KKXK motifs within the C-terminal NLS/NoLS region of PAK1IP1 are essential for nucleolar and nuclear localization of the protein. To test if PAK1IP1 was responsive to nucleolar stress, HeLa cells were treated with chemotherapeutic agents fluorouracil and low dose actinomycin D. The protein level of PAK1IP1 was markedly increased in a dose-dependent fashion, but the mRNA of PAK1IP1 had no significant change. Ribosomal stress has been reported to induce the cell cycle arrest. We then examined whether the expression of PAK1IP1 was regulated during the cell cycle. HeLa cells were synchronized by double thymidine treatment, and then the cells were released. We found that PAK1IP is highly expressed in G0/G1 phase and down-regulated in S and G2/M phase of the cell cycle. These results suggest that the expression of PAK1IP1 is ribosomal stress-induced and cell cycle-regulated.B. PAK1IP1 regulates cell cycle progression through modulation of p53 stabilityTo assess whether PAK1IP1 affects cell proliferation, we employed a modified lentiviral-based pLL3.7 vector system for the expression of GFP-PAK1IP1 or its mutant forms. U2OS cells were infected with control, wild type or mutant PAK1IP1 lentifiviruses, respectively, and the cell growth curve, MTS and colony formation were analyzed. It is shown that wild type PAK1IP1 and PAK1IP1-mNoLS mutant dramatically inhibited cell proliferation, whereas PAK1IP1-dNLS mutant did not show any effect. Of note, the inhibition effect of wild type PAK1IP1 was stronger than that of the PAK1IP1-mNoLS mutant. These results demonstrate that ectopic expression of PAK1IP1 can inhibit cell proliferation and this inhibition relies on its nuclear localization.To determine whether the inhibition of cell proliferation by PAK1IP1 is associated with p53 by using p53-proficientand p53-deficient cell lines for colony formation assays. Overexpression of PAK1IP1 and PAK1IP1-mNoLS inhibited colony formation in p53-containing, but not p53-deficient cells.To identify whether overexpression of PAK1IP1 is involved in Gl/S progression, the 5-bromodeoxyuridine (BrdU) incorporation assays. Overexpression of PAK1IP1 and PAK1IP1-mNoLS significantly reduced the number of BrdU positive cells, suggesting that PAK1IP1 indeed inhibits cell proliferation via reduction of DNA .synthesis. Through siRNA-mediated p53 knockdown and flow cytometry cell sorting assay (FACS) analysis, we found that significantly more cells that expressed GFP-PAK1IP1 were accumulated at G1 phase than that of control. However, this result was found in p53-knockdown and p53-difficient cells.To determine whether PAK1IP1 induces p53-dependent G1 arrest by directly targeting to p53, we analyzed p53 levels after overexpression of PAK1IP1 by immunofluorescence staining and Western blot analysis in U2OS cells. Overexpression of PAK1IP1 or PAK1IP1-mNoLS but not the PAK1IP1-dNLS induces p53 and its targets. To investigate how PAK1IP1 induces p53 levels, the half-life of p53 was examined in PAK1IP1 or vector lentivirus infected U2OS cells. The half-life of the p53 protein was significantly extended from half an hour in control cells to a few hours in PAK1IP1 infected cells. These results suggest that high levels of PAK1IP1 can stabilize p53 levels. Next, we found that both endogenous and exogenous PAK1IP1 interacts with MDM2, and PAK1IP1 can stabilize p53 by inhibiting MDM2-mediated p53 ubiquitination and degradation.C. Knocking down of PAK1IP1 induces cell cycle arrest and impairs rRNA biogenesisTo determine the physiological relevance of PAK1IP1 in the regulation of cell proliferation, we employed a lentiviral based RNA interference system and designed two shRNAs of PAK1IP1. Both the mRNA and protein level of endogenous PAK1IP1 were efficiently ablated when shRNA2 was introduced into the cells. Surprisingly, knockdown of endogenous PAK1IP1 also inhibited cell proliferation by cell growth, cell viability and colony formation assays in HeLa cells, and we also found that this inhibition was more apparent in p53-proficient cells than in p53-deficient cells, Suggesting that knockdown of PAK1IP1 may also induce p53-dependent cell growth arrest. We analyzed p53 levels after knockdown of PAK1IP1 by Western blot analysis. Knockdown of endogenous PAK1IP1 induced p53, and drastically induced cell cycle arrest in the G1 phase.Next, we determined whether the knockdown of PAK1IP1 by siRNA could activate the ribosomal protein-MDM2-p53 pathway. We found that the PAK1IP1 knockdown indeed increased the free ribosomal proteins L5 and L11 through sucrose gradient centrifugation, and enhanced the interaction between MDM2 and L5 or L11 with co-immunoprecipitation. These results revealed that knockdown of PAK1IP1 may induce ribosomal stress. With the sucrose gradient analysis of pre-ribosomal ribonucleoprotein complexes, we found that silencing of PAK1IP1 reduced the 60S ribosome assembly. And we also identified that it inhibited the 28S rRNA processing through Northern blot assay with 32p labeled oligonucleotide probes and the pulse-chase analysis of L-[methyl-3H] methionine. Suggesting that knockdown of PAK1IP1 induces ribosomal tress for the ribosome assembly reduction, and lead to cell proliferation inhibition.Collectively, our study as presented here identified a novel nucleolar protein PAKIIPI which could be induced by nucleolar stress and regulated cell cycle progression via p53-MDM2 pathway. These findings are very interesting, highlighting the importance of the fine balance of this nucleolar protein level for cell proliferation, as any of its off-balance would cause a stress leading to p53 activation and cell growth arrest. This may not only explain what an important role PAK1IP1 plays in the evolution, but also provide a good evidence for the p53-MDM2 pathway response to nucleolar stress.