Direct roles of the signaling kinase RSK2 in Cdc25C activation during Xenopus oocyte maturation.

The induction of M phase in eukaryotic cell cycles requires robust activation of Cdc2/cyclin B by Cdc25, which itself is robustly activated by serine/threonine phosphorylations. Although multiple protein kinases that directly activate Cdc25C have been identified, whether the combination of different primary phosphorylations of Cdc25C is sufficient to fully activate Cdc25C has not been determined. By analyzing the GST-Cdc25C phosphorylating activity in Xenopus egg extracts, we previously defined roles of MAPK and Cdc2/cyclin B in partially activating Cdc25C and predicted the presence of another major Cdc25C-activating kinase. In this study, we demonstrate that this missing kinase is RSK2, which phosphorylates three sites in Cdc25C and also partially activates Cdc25C. However, the phosphorylations catalyzed by MAPK, Cdc2, and RSK2 fail to fully activate Cdc25C, suggesting that additional biochemical events are required to fully activate this key cell cycle regulator.

Disruption of the dynamic sub-cellular localization of the Xenopus tumorhead protein causes embryonic lethality at the early gastrula transition.

The Xenopus laevis tumorhead (TH) protein, a positive regulator of cell proliferation during embryogenesis, shuttles from the cell periphery into the nucleus during embryogenesis. In these studies, we performed a detailed analysis of TH's subcellular localization pattern to characterize its dynamic behavior. We found that TH exhibits distinct patterns of localization in different germ layers. At the blastula stage, TH is present in the apical cell periphery of prospective mesodermal and ectodermal cells. At the gastrula stage, TH is distributed throughout the entire cytoplasm of prospective mesodermal and ectodermal cells, whereas it shows nuclear localization in presumptive endodermal cells. TH moves into the nucleus of mesodermal and ectodermal cells during the neurula and early tailbud stages. To understand if TH is regulated by changes in its subcellular localization, we used a TH mutant containing signals for farnesylation and palmitoylation to tether the protein to the plasma membrane. Ubiquitous overexpression of this mutant causes embryonic lethality at the early gastrula transition. Further examination using TUNEL assays indicated that wild-type TH overexpression induces apoptosis during gastrulation, and that this effect is exacerbated by the overexpression of the membrane-bound TH mutant. Taken together, our results suggest that changes in the sub-cellular localization of the TH protein are important for its function because blocking the nuclear translocation of overexpressed TH increases apoptosis and causes embryos to die. Our data also suggest that TH plays a role outside the nucleus when it is present at the cell periphery.

Identification of TH-B, a new oligomeric variant of the Xenopus morphogenetic factor, tumorhead.

The Xenopus laevis gene tumorhead (TH) is a regulator of cell proliferation of the ectodermal germ layer during embryonic development. TH overexpression results in increased cell proliferation within the developing ectoderm, causing an expansion of the neural plate. Conversely, loss of TH function results in inhibition of proliferation of ectodermal cells. Embryos with altered levels of TH protein are unable to express neural differentiation markers, indicating that the effect of TH in proliferation is linked with differentiation in the nervous system. To date, the molecular mechanism by which TH affects cell proliferation during embryogenesis is unknown. We have utilized the yeast two-hybrid system to identify protein partners of TH that could lead us to define the mechanism or pathway through which TH functions. Using this assay we have identified a new variant of TH designated TH-B, as a potential protein partner of the original TH, now referred to as TH-A. The sequence for TH-B was found to be 85% identical at the amino acid level to the TH-A sequence. Further characterization of the TH-B variant using RT-PCR indicates that it is expressed ubiquitously throughout development from early cleavage stages until at least the tadpole stage. TH-B association with TH-A was confirmed in co-immnoprecipitation studies in Xenopus, indicating that the two variants may function as an oligomer in vivo. These studies reveal the presence of an isoform of TH that may possess novel functional capabilities.

RSK promotes prostate cancer progression in bone through ING3, CKAP2, and PTK6-mediated cell survival.

UNLABELLED: Prostate cancer has a proclivity to metastasize to bone. The mechanism by which prostate cancer cells are able to survive and progress in the bone microenvironment is not clear. Identification of molecules that play critical roles in the progression of prostate cancer in bone will provide essential targets for therapy. Ribosomal S6 protein kinases (RSK) have been shown to mediate many cellular functions critical for cancer progression. Whether RSK plays a role in the progression of prostate cancer in bone is unknown. IHC analysis of human prostate cancer specimens showed increased phosphorylation of RSK in the nucleus of prostate cancer cells in a significant fraction of human prostate cancer bone metastasis specimens, compared with the primary site or lymph node metastasis. Expression of constitutively active myristylated RSK in C4-2B4 cells (C4-2B4/RSK) increased their survival and anchorage-independent growth compared with C4-2B4/vector cells. Using an orthotopic bone injection model, it was determined that injecting C4-2B4/RSK cells into mouse femurs enhanced their progression in bone compared with control cells. In PC3-mm2 cells, knockdown of RSK1 (RPS6KA1), the predominant RSK isoform, but not RSK2 (RPS6KA2) alone, decreased anchorage-independent growth in vitro and reduced tumor progression in bone and tumor-induced bone remodeling in vivo. Mechanistic studies showed that RSK regulates anchorage-independent growth through transcriptional regulation of factors that modulate cell survival, including ING3, CKAP2, and PTK6. Together, these data provide strong evidence that RSK is an important driver in prostate cancer progression in bone. IMPLICATIONS: RSK, an important driver in prostate cancer progression in bone, has promising potential as a therapeutic target for prostate cancer bone metastasis.

Perspectives on the origin of microfilaments, microtubules, the relevant chaperonin system and cytoskeletal motors--a commentary on the spirochaete origin of flagella.

The origin of cytoskeleton and the origin of relevant intracellular transportation system are big problems for understanding the emergence of eukaryotic cells. The present article summarized relevant information of evidences and molecular traces on the origin of actin, tubulin, the chaperonin system for folding them, myosins, kinesins, axonemal dyneins and cytoplasmic dyneins. On this basis the authors proposed a series of works, which should be done in the future, and indicated the ways for reaching the targets. These targets are mainly: 1) the reconstruction of evolutionary path from MreB protein of archaeal ancestor of eukaryotic cells to typical actin; 2) the finding of the MreB or MreB-related proteins in crenarchaea and using them to examine J. A. Lake's hypothesis on the origin of eukaryote from "eocytes" (crenarchaea); 3) the examinations of the existence and distribution of cytoskeleton made of MreB-related protein within coccoid archaea, especially in amoeboid archaeon Thermoplasm acidophilum; 4) using Thermoplasma as a model of archaeal ancestor of eukaryotic cells; 5) the searching for the homolog of ancestral dynein in present-day living archaea. During the writing of this article, Margulis' famous spirochaete hypothesis on the origin of flagella and cilia was unexpectedly involved and analyzed from aspects of tubulins, dyneins and spirochaetes. Actually, spirochaete cannot be reasonably assumed as the ectosymbiotic ancestor of eukaryotic flagella and cilia, since their swing depends upon large amount of bacterial flagella beneath the flexible outer wall, but not depends upon their intracellular tubules and the assumed dyneins. In this case, if they had "evolved" into cilia and lost their bacterial flagella, they would immediately become immobile! In fact, tubulin and dynein-like proteins have not been found in any spirochaete.

Dissecting the M phase-specific phosphorylation of serine-proline or threonine-proline motifs.

M phase induction in eukaryotic cell cycles is associated with a burst of protein phosphorylation, primarily at serine or threonine followed by proline (S/TP motif). The mitotic phosphoprotein antibody MPM-2 recognizes a significant subset of mitotically phosphorylated S/TP motifs; however, the required surrounding sequences of and the key kinases that phosphorylate these S/TP motifs remain to be determined. By mapping the mitotic MPM-2 epitopes in Xenopus Cdc25C and characterizing the mitotic MPM-2 epitope kinases in Xenopus oocytes and egg extracts, we have determined that phosphorylation of TP motifs that are surrounded by hydrophobic residues at both -1 and +1 positions plays a dominant role in M phase-associated burst of MPM-2 reactivity. Although mitotic Cdk and MAPK may phosphorylate subsets of these motifs that have a basic residue at the +2 position and a proline residue at the -2 position, respectively, the majority of these motifs that are preferentially phosphorylated in mitosis do not have these features. The M phase-associated burst of MPM-2 reactivity can be induced in Xenopus oocytes and egg extracts in the absence of MAPK or Cdc2 activity. These findings indicate that the M phase-associated burst of MPM-2 reactivity represents a novel type of protein phosphorylation in mitotic regulation.

Alternative polyadenylation produces two major transcripts of Alix.

The mammalian adaptor protein Alix participates in multiple cellular processes. Since mouse Alix cDNA detects two distinct transcripts of approximately 3.5 and approximately 7.0 kb in various mouse tissues, it is possible that there exist isoforms of Alix protein that perform varied biological functions. In this study, we first demonstrate that four different anti-Alix monoclonal antibodies immunoblot the single Alix protein in nine different mouse tissues. We then show that the two transcripts of 3.2 and 6.4 kb are widely expressed in various human tissues and cell lines. These two transcripts are generated from the same Alix gene localizing at 3p22.3 via alternative polyadenylation, thus containing an identical open reading frame. However, the 3.2-kb transcript is much more active in translation than the 6.4-kb transcript in a randomly selected cell line. These results eliminate the possibility that the two transcript variants encode different isoforms of Alix protein and suggest that alternative polyadenylation is one of the mechanisms controlling Alix protein expression.

Tumorhead distribution to cytoplasmic membrane of neural plate cells is positively regulated by Xenopus p21-activated kinase 1 (X-PAK1).

Tumorhead (TH) regulates neural plate cell proliferation during Xenopus early development, and gain or loss of function prevents neural differentiation. TH shuttles between the nuclear and cytoplasmic/cortical cell compartments in embryonic cells. In this study, we show that subcellular distribution of TH is important for its functions. Targeting TH to the cell cortex/membrane potentiates a TH gain of function phenotype and results in neural plate expansion and inhibition of neuronal differentiation. We have found that TH subcellular localization is regulated, and that its shuttling between the nucleus and the cell cortex/cytoplasm is controlled by the catalytic activity of p21-activated kinase, X-PAK1. The phenotypes of embryos that lack, or have excess, X-PAK1 activity mimic the phenotypes induced by loss or gain of TH functions, respectively. We provide evidence that X-PAK1 is an upstream regulator of TH and discuss potential functions of TH at the cell cortex/cytoplasmic membrane and in the nucleus.

New symbiotic hypothesis on the origin of eukaryotic flagella.

The origin of eukaryotic flagella has long been a mystery. Here we review the possibility that flagella sprouted evolutionarily from the eukaryotic cell proper seems very unlikely because it is hard to imagine what function and benefit in natural selection the flagella would have provided to the cells when they first emerged as simple buds. Lynn Margulis' 1970 spirochete hypothesis, though popular still, has never been confirmed. Moreover, the absence of tubulin and axonemal dynein in the spirochetes and the incapability of the bacterial and eukaryotic membranes' making a continuum now suggest that the hypothesis is outdated. Tubulin genes were recently identified in a new bacteria division, verrucomicrobia, and microtubules have also been found in one of these species, epixenosomes, the defensive ectosymbionts. On the basis of these data, we propose a new symbiotic hypothesis: that the mid-ancestor of eukaryotic cells obtained epixenosomelike verrucomicrobia as defensive ectosymbionts and the ectosymbionts later became endosymbiotic. They still, however, protruded from the surface of their host to play their role. Later, many genes were lost or incorporated into the host genome. Finally, the genome, the bacterial membrane, and the endosymbiotic vesicle membrane were totally lost, and fingerlike protrusions with microtubules formed. As the cells grew larger, the defensive function of the protrusions eventually weakened and then vanished. Some of the protrusions took on a new role in cell movement, which led them to evolve into flagella. The key step in this process was that the dynein obtained from the host evolved into axonemal dyneins, attaching onto the microtubules and forming motile axonemes. Our hypothesis is unproven, but it offers a possible explanation that is consistent with current scientific thought. We hope that our ideas will stimulate additional studies on the origin of eukaryotic flagella and on investigations of verrucomicrobia. Whether such studies confirm, refine, or replace our hypothesis, they should nevertheless further our understanding of the origin of eukaryotic cells.

The Xenopus laevis morphogenetic factor, tumorhead, causes defects in polarized growth and cytokinesis in the fission yeast, Schizosaccharomyces pombe.

Tumorhead (TH) is a maternally expressed gene product that regulates neural tube morphogenesis in the amphibian, Xenopus laevis. Here we describe the effects of TH expression in the rod-shaped fission yeast, Schizosaccharomyces pombe. Expression of TH in S. pombe resulted in severe morphological defects, including ovoid, bottle-shaped, and enlarged cells. Multi-septated cells were also observed in TH expressing cultures, indicating that TH is inhibitory to a process required for the completion of cytokinesis. TH expression caused significant actin and microtubule cytoskeletal defects, including depolarization of the cortical F-actin cytoskeleton and increased microtubule formation. Immunostaining experiments showed that TH is localized to the cell cortex, cell tips, and septum in S. pombe cells. Localization of TH to the cell cortex was dependent on the S. pombe PAK homolog, Shk1. Moreover, TH expression was inhibitory to the growth of a mutant defective in Shk1 function, suggesting that TH may interact with a component(s) of a PAK-mediated morphogenetic regulatory pathway in S. pombe. Taken together, our findings demonstrate that S. pombe may be a useful model organism for identifying potential TH interacting factors.

Difference in the maternal and zygotic contributions of tumorhead on embryogenesis.

Tumorhead (TH) is a maternally expressed gene in Xenopus laevis, that when overexpressed, increased proliferation of ectodermal derivatives and inhibited neural and epidermal differentiation. However, injection of anti-TH antibodies inhibited cleavage of all blastomeres, not only those contributing to the ectoderm. The injection of TH morpholino antisense oligonucleotide (TH-MO), which inhibits translation of TH mRNA, did not affect early cleavage but inhibited cell division in both the neural field and epidermis. This was accompanied by the inhibition of neural and epidermal markers. TH-MO did not affect the formation and differentiation of mesoderm and endoderm derivatives. Our overexpression and loss-of-function studies demonstrated that TH plays an important role in differentiation of the ectoderm by regulating cell proliferation. They also supported the conclusion that the maternal component of TH may affect the cell cycle in all cells, while the zygotic component has a germ layer-specific effect on the ectoderm.