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Overexpression or activation of oncogenes or loss of tumor-suppressor genes can induce cellular senescence as a defense mechanism against tumor development, thereby maintaining cellular homeostasis. However, cancer cells can circumvent this senescent state and continue to spread. Myosin light chain kinase (MLCK) is downregulated in many breast cancers. Here we report that downregulation of MLCK in normal breast epithelial cells induces a senescence-associated secretory phenotype and stimulates migration. The reduction of MLCK results in increased p21 Cip1 expression, dependent on p53 and the AKT-mammalian target of rapamycin pathway. Subsequently, p21 Cip1 promotes the secretion of soluble ICAM-1, IL-1α, IL-6 and IL-8, thereby enhancing collective cell migration in a non-cell-autonomous manner. These findings provide new mechanistic insights into the role of MLCK in cellular senescence and cancer progression.

Using MDA-MB-231 cells as a model of triple negative breast cancer (TNBC) and its metastatic sub-cell lines that preferentially metastasize to lung, bone or brain, we found that the mRNA and protein levels of fibronectin (FN) are increased in MDA-MB-231 cells and its lung metastatic derivative, when cultivated in three-dimensional (3D) suspension cultures. The increase of FN expression in 3D was dependent on p38 mitogen-activated protein kinase (MAPK) because it was prevented by treatment of cells with SB203580, an inhibitor of p38MAPK. The up-regulated FN was converted into fibrils, and it enhanced cell spreading when cells cultured in 3D were transferred to two-dimensional (2D) culture. The arginine-glycine-aspartate (RGD) peptides and siRNAs targeting of integrin β-5 inhibited spreading of cells regardless of the presence of FN on 2D culture dishes. In addition, the levels of phosphorylated Src were found to be increased in 3D and the treatment of cells with SU6656, an inhibitor of Src, decreased the rate of cell spreading on FN. Collectively, these studies demonstrate that increased cellular FN in 3D suspension culture facilitates cancer cell attachment and spreading via integrin β-5 and Src, suggesting that the increased FN promotes initial attachment of cancer cells to secondary organs after circulation during metastasis.

p90 ribosomal S6 kinase (RSK1) is an effector of both Ras/MEK/MAPK and PI3K/PDK1 pathways. We present evidence that RSK1 drives p27 phosphorylation at T198 to increase RhoA-p27 binding and cell motility. RSK1 activation and p27pT198 both increase in early G₁. As for many kinase-substrate pairs, cellular RSK1 coprecipitates with p27. siRNA to RSK1 and RSK1 inhibition both rapidly reduce cellular p27pT198. RSK1 overexpression increases p27pT198, p27-cyclin D1-Cdk4 complexes, and p27 stability. Moreover, RSK1 transfectants show mislocalization of p27 to cytoplasm, increased motility, and reduced RhoA-GTP, phospho-cofilin, and actin stress fibers, all of which were reversed by shRNA to p27. Phosphorylation by RSK1 increased p27pT198 binding to RhoA in vitro, whereas p27T157A/T198A bound poorly to RhoA compared with WTp27 in cells. Coprecipitation of cellular p27-RhoA was increased in cells with constitutive PI3K activation and increased in early G₁. Thus T198 phosphorylation not only stabilizes p27 and mislocalizes p27 to the cytoplasm but also promotes RhoA-p27 interaction and RhoA pathway inhibition. These data link p27 phosphorylation at T198 and cell motility. As for other PI3K effectors, RSK1 phosphorylates p27 at T198. Because RSK1 is also activated by MAPK, the increased cell motility and metastatic potential of cancer cells with PI3K and/or MAPK pathway activation may result in part from RSK1 activation, leading to accumulation of p27T198 in the cytoplasm, p27:RhoA binding, inhibition of RhoA/Rock pathway activation, and loss of actomyosin stability.

The opposing effects of SF2/ASF and heterogeneous nuclear ribonucleoprotein (hnRNP) A1 influence alternative splicing in vitro. SF2/ASF or hnRNP A1 complementary DNAs were transiently overexpressed in HeLa cells, and the effect on alternative splicing of several cotransfected reporter genes was measured. Increased expression of SF2/ASF activated proximal 5′ splice sites, promoted inclusion of a neuron-specific exon, and prevented abnormal exon skipping. Increased expression of hnRNP A1 activated distal 5′ splice sites. Therefore, variations in the intracellular levels of antagonistic splicing factors influence different modes of alternative splicing in vivo and may be a natural mechanism for tissue-specific or developmental regulation of gene expression.

Mutations in the Schizosaccharomyces pombe cdc8 gene impair cytokinesis. Here we clone cdc8+ and find that it encodes a novel tropomyosin. Gene disruption results in lethal arrest of the cell cycle, but spore germination, cell growth, DNA replication and mitosis are all unaffected. Haploid cdc8 gene disruptants are rescued by expression of a fibroblast tropomyosin complementary DNA. Immunofluorescence microscopy of wild type and cdc8 gene disruptants indicates that cdc8 tropomyosin is present in two distinct cellular distributions: in dispersed patches, and during cytokinesis as a transient medial band. Collectively these results indicate that cdc8 tropomyosin has a specialized role which, we suggest, is to form part of the F-actin contractile ring at cytokinesis. These results establish the basis for further genetic studies of cytokinesis and of contractile protein function in S. pombe.

Transformation of cells in tissue culture results in a variety of cellular changes including alterations in cell growth, adhesiveness, motility, morphology, and organization of the cytoskeleton. Morphological and cytoskeletal changes are perhaps the most readily apparent features of transformed cells. Although a number of studies have documented a decrease in the expression of specific tropomyosin (TM) isoforms in transformed cells, it remains to be determined if the suppression of TM synthesis is essential in the establishment and maintenance of the transformed phenotype. To address the roles of different TM isoforms in transformed cells we have examined the effects of expressing specific TM isoforms in transformed cells using a Kirsten virus-transformed cell line (ATCC NRK 1569) as a model system. In contrast to normal fibroblasts, the NRK 1569 cells contain reduced levels of TM-1 and undetectable levels of TM-2 and TM-3. These cells have a rounded morphology and are devoid of stress fibers. Employing expression plasmids for TM-2 and TM-3, stable cell lines were established from the NRK 1569 cells that express these isoforms individually. We demonstrate that expression of TM-2 or TM-3 leads to increased cell spreading accompanied by the formation of identifiable microfilament bundles, as well as significant restoration of well-defined vinculin-containing focal adhesion plaques, although expression of each isoform exhibited distinct properties. In addition, cells expressing TM-2, but not TM-3, exhibited contact-inhibited cell growth and a requirement for serum.

ACTIN is a cytoskeletal protein which is highly conserved across eukaryotic phyla. Actin filaments, in association with a family of myosin motor proteins, are required for cellular motile processes as diverse as vesicle transport, cell locomotion and cytokinesis 1,2 . Many organisms have several closely related actin isoforms 3,4 . In addition to conventional actins, yeasts contain actin-related proteins that are essential for viability 5,6 . We show here that vertebrates also contain an actin-related protein (actin-RPV). Actin-RPV is a major component of the dynactin complex, an activator of dynein-driven vesicle movement 7,8 , indicating that unlike conventional actins which work in conjunction with myosin motors, actin-RPV may be involved in cytoplasmic movements via a microtubule-based system.

Actins are a family of highly conserved proteins that are ubiquitously found among eukaryotic organisms. All actins that have previously been identified, including those of animals, plants, fungi, and protozoa, are 374-376 amino acids long and exhibit at least 70% amino acid sequence identity when compared with one another. We have cloned a gene from the fission yeast Schizosaccharomyces pombe that encodes a distantly related member of the actin protein family, herein referred to as act2. In contrast to all other actins, the derived amino acid sequence reveals that act2 is 427 residues long and exhibits only 35-40% identity to actins, including act1 from Sch. pombe. Comparison to the known x-ray crystallographic structure of rabbit skeletal muscle actin indicates that the ATP and divalent metal ion binding sites are largely conserved in act2, while regions involved in actin-actin and actin-myosin interactions are relatively divergent. Disruption of the act2 gene demonstrated that this gene encodes a function essential for germination of haploid spores. These findings indicate that while act2 and act1 are related proteins, they appear to have distinct functions. In addition, they demonstrate that the actin protein family is more diverse than was previously thought.

In this study we report on the developmental and regional expression of two brain-specific isoforms of tropomyosin, TMBr-1 and TMBr-3, that are generated from the rat α-tropomyosin gene via the use of alternative promoters and alternative RNA splicing. Western blot analysis using an exon-specific peptide polyclonal antibody revealed that the two isoforms are differentially expressed in development with TMBr-3 appearing in the embryonic brain at 16 days of gestation, followed by the expression of TMBr-1 at 20 days after birth. TMBr-3 was detected in all brain regions examined, whereas TMBr-1 was detected predominantly in brain areas that derive from the prosencephalon. Immunocytochemical studies on mixed primary cultures made from rat embryonic midbrain indicate that expression of the brain-specific epitope is restricted to neurons. The developmental pattern and neuronal localization of these forms of tropomyosin suggest that these isoforms have a specialized role in the development and plasticity of the nervous system.

p90 ribosomal S6 kinase (RSK1) is an effector of both Ras/MEK/MAPK and PI3K/PDK1 pathways. We present evidence that RSK1 drives p27 phosphorylation at T198 to increase RhoA-p27 binding and cell motility. RSK1 activation and p27pT198 both increase in early G 1 . As for many kinase–substrate pairs, cellular RSK1 coprecipitates with p27. siRNA to RSK1 and RSK1 inhibition both rapidly reduce cellular p27pT198. RSK1 overexpression increases p27pT198, p27-cyclin D1-Cdk4 complexes, and p27 stability. Moreover, RSK1 transfectants show mislocalization of p27 to cytoplasm, increased motility, and reduced RhoA-GTP, phospho-cofilin, and actin stress fibers, all of which were reversed by shRNA to p27. Phosphorylation by RSK1 increased p27pT198 binding to RhoA in vitro, whereas p27T157A/T198A bound poorly to RhoA compared with WTp27 in cells. Coprecipitation of cellular p27-RhoA was increased in cells with constitutive PI3K activation and increased in early G 1 . Thus T198 phosphorylation not only stabilizes p27 and mislocalizes p27 to the cytoplasm but also promotes RhoA-p27 interaction and RhoA pathway inhibition. These data link p27 phosphorylation at T198 and cell motility. As for other PI3K effectors, RSK1 phosphorylates p27 at T198. Because RSK1 is also activated by MAPK, the increased cell motility and metastatic potential of cancer cells with PI3K and/or MAPK pathway activation may result in part from RSK1 activation, leading to accumulation of p27T198 in the cytoplasm, p27:RhoA binding, inhibition of RhoA/Rock pathway activation, and loss of actomyosin stability.