Non-phosphorylatable cyclin D1 mutant potentiates endometrial hyperplasia and drives carcinoma with Pten loss.

Cyclin D1 is a regulatory subunit of -Cyclin Dependent Kinases 4 and 6 (CDK4/6) and regulates progression from G1 to S phase of the cell cycle. Dysregulated cyclin D1-CDK4/6 contributes to abnormal cell proliferation and tumor development. Phosphorylation of threonine 286 of cyclin D1 is necessary for ubiquitin-dependent degradation. Non-phosphorylatable cyclin D1 mutants are stabilized and concentrated in the nucleus, contributing to genomic instability and tumor development. Studies investigating the tumor-promoting functions of cyclin D1 mutants have focused on the use of artificial promoters to drive the expression which unfortunately may not accurately reflect tumorigenic functions of mutant cyclin D1 in cancer development. We have generated a conditional knock-in mouse model where cyclin D1T286A is expressed under the control of its endogenous promoter following Cre-dependent excision of a lox-stop-lox sequence. Acute expression of cyclin D1T286A following tamoxifen-inducible Cre recombinase triggers inflammation, lymphocyte abnormality and ultimately mesenteric tumors in the intestine. Tissue-specific expression of cyclin D1T286A in the uterus and endometrium cooperates with Pten loss to drive endometrial hyperplasia and cancer. Mechanistically, cyclin D1T286A mutant activates NF-κB signaling, augments inflammation, and contributes to tumor development. These results indicate that mutation of cyclin D1 at threonine 286 has a critical role in regulating inflammation and tumor development.

A two-track model for the spatiotemporal coordination of bacterial septal cell wall synthesis revealed by single-molecule imaging of FtsW.

Synthesis of septal peptidoglycan (sPG) is crucial for bacterial cell division. FtsW, an indispensable component of the cell division machinery in all walled bacterial species, was recently identified in vitro as a peptidoglycan glycosyltransferase (PGTase). Despite its importance, the septal PGTase activity of FtsW has not been demonstrated in vivo. How its activity is spatiotemporally regulated in vivo has also remained elusive. Here, we confirmed FtsW as an essential septum-specific PGTase in vivo using an N-acetylmuramic acid analogue incorporation assay. Next, using single-molecule tracking coupled with genetic manipulations, we identified two populations of processively moving FtsW molecules: a fast-moving population correlated with the treadmilling dynamics of the essential cytoskeletal FtsZ protein and a slow-moving population dependent on active sPG synthesis. We further identified that FtsN, a potential sPG synthesis activator, plays an important role in promoting the slow-moving population. Our results suggest a two-track model, in which inactive sPG synthases follow the 'Z-track' to be distributed along the septum and FtsN promotes their release from the Z-track to become active in sPG synthesis on the slow 'sPG-track'. This model provides a mechanistic framework for the spatiotemporal coordination of sPG synthesis in bacterial cell division.

Roles of the DedD Protein in Escherichia coli Cell Constriction.

Two key tasks of the bacterial septal-ring (SR) machinery during cell constriction are the generation of an inward-growing annulus of septal peptidoglycan (sPG) and the concomitant splitting of its outer edge into two layers of polar PG that will be inherited by the two new cell ends. FtsN is an essential SR protein that helps trigger the active constriction phase in Escherichia coli by inducing a self-enhancing cycle of processes that includes both sPG synthesis and splitting and that we refer to as the sPG loop. DedD is an SR protein that resembles FtsN in several ways. Both are bitopic inner membrane proteins with small N-terminal cytoplasmic parts and larger periplasmic parts that terminate with a SPOR domain. Though absence of DedD normally causes a mild cell-chaining phenotype, the protein is essential for division and survival of cells with limited FtsN activity. Here, we find that a small N-terminal portion of DedD (NDedD; DedD1-54) is required and sufficient to suppress ΔdedD-associated division phenotypes, and we identify residues within its transmembrane domain that are particularly critical to DedD function. Further analyses indicate that DedD and FtsN act in parallel to promote sPG synthesis, possibly by engaging different parts of the FtsBLQ subcomplex to induce a conformation that permits and/or stimulates the activity of sPG synthase complexes composed of FtsW, FtsI (PBP3), and associated proteins. We propose that, like FtsN, DedD promotes cell fission by stimulating sPG synthesis, as well as by providing positive feedback to the sPG loop.IMPORTANCE Cell division (cytokinesis) is a fundamental biological process that is incompletely understood for any organism. Division of bacterial cells relies on a ring-like machinery called the septal ring or divisome that assembles along the circumference of the mother cell at the site where constriction eventually occurs. In the well-studied bacterium Escherichia coli, this machinery contains over 30 distinct proteins. We identify functionally important parts of one of these proteins, DedD, and present evidence supporting a role for DedD in helping to induce and/or sustain a self-enhancing cycle of processes that are executed by fellow septal-ring proteins and that drive the active constriction phase of the cell division cycle.

A protease storm cleaves a cell-cell adhesion molecule in cancer: multiple proteases converge to regulate PTPmu in glioma cells.

Cleavage of the cell-cell adhesion molecule, PTPµ, occurs in human glioblastoma multiforme brain tumor tissue and glioma cell lines. PTPµ cleavage is linked to increased cell motility and growth factor independent survival of glioma cells in vitro. Previously, PTPµ was shown to be cleaved by furin in the endoplasmic reticulum to generate membrane associated E- (extracellular) and P- (phosphatase) subunits, and by ADAMs and the gamma secretase complex at the plasma membrane. We also identified the presence of additional extracellular and intracellular PTPµ fragments in brain tumors. We set out to biochemically analyze PTPµ cleavage in cancer cells. We determined that, in addition to the furin-processed form of PTPµ, a pool of 200 kDa full-length PTPµ exists at the plasma membrane that is cleaved directly by ADAM to generate a larger shed form of the PTPµ extracellular segment. Notably, in glioma cells, full-length PTPµ is also subject to calpain cleavage, which generates novel PTPµ fragments not found in other immortalized cells. We also observed glycosylation and phosphorylation differences in the cancer cells. Our data suggest that an additional serine protease also contributes to PTPµ shedding in glioma cells. We hypothesize that a "protease storm" occurs in cancer cells whereby multiple proteases converge to reduce the presence of cell-cell adhesion molecules at the plasma membrane and to generate protein fragments with unique biological functions. As a consequence, the "protease storm" could promote the migration and invasion of tumor cells.

Mapping the mechanome of live stem cells using a novel method to measure local strain fields in situ at the fluid-cell interface.

During mesenchymal condensation, the initial step of skeletogenesis, transduction of minute mechanical forces to the nucleus is associated with up or down-regulation of genes, ultimately resulting in formation of the skeletal template and appropriate cell lineage commitment. The summation of these biophysical cues affects the cell's shape and fate. Here, we predict and measure surface strain, in live stem cells, in response to controlled delivery of stresses, providing a platform to direct short-term structure--function relationships and long-term fate decisions. We measure local strains on stem cell surfaces using fluorescent microbeads coated with Concanavalin A. During delivery of controlled mechanical stresses, 4-Dimensional (x,y,z,t) displacements of the bound beads are measured as surface strains using confocal microscopy and image reconstruction. Similarly, micro-particle image velocimetry (µ-piv) is used to track flow fields with fluorescent microspheres. The measured flow velocity gradient is used to calculate stress imparted by fluid drag at the surface of the cell. We compare strain measured on cell surfaces with those predicted computationally using parametric estimates of the cell's elastic and shear modulus. Finally, cross-correlating stress--strain data to measures of gene transcription marking lineage commitment enables us to create stress--strain--fate maps, for live stem cells in situ. The studies show significant correlations between live stem cell stress--strain relationships and lineage commitment. The method presented here provides a novel means to probe the live stem cell's mechanome, enabling mechanistic studies of the role of mechanics in lineage commitment as it unfolds.

Protein tyrosine phosphatase mu regulates glioblastoma cell growth and survival in vivo.

Glioblastoma multiforme (GBM) is the most lethal primary brain tumor. Extensive proliferation and dispersal of GBM tumor cells within the brain limits patient survival to approximately 1 year. Hence, there is a great need for the development of better means to treat GBM. Receptor protein tyrosine phosphatase (PTP)µ is proteolytically cleaved in GBM to yield fragments that promote dispersal of GBM cells. While normal brain tissue retains expression of full-length PTPµ, low-grade human astrocytoma samples have varying amounts of full-length PTPµ and cleaved PTPµ. In the highest-grade astrocytomas (i.e., GBM), PTPµ is completely proteolyzed into fragments. We demonstrate that short hairpin RNA mediated knockdown of full-length PTPµ and PTPµ fragments reduces glioma cell growth and survival in vitro. The reduction in growth and survival following PTPµ knockdown is enhanced when cells are grown in the absence of serum, suggesting that PTPµ may regulate autocrine signaling. Furthermore, we show for the first time that reduction of PTPµ protein expression decreases the growth and survival of glioma cells in vivo using mouse xenograft flank and i.c. tumor models. Inhibitors of PTPµ could be used to reduce the growth and survival of GBM cells in the brain, representing a promising therapeutic target for GBM.

Cadherin-11, a marker of the mesenchymal phenotype, regulates glioblastoma cell migration and survival in vivo.

Glioblastoma multiforme (GBM) is the most malignant and lethal form of astrocytoma. The GBM patient survival time of approximately 1 year necessitates the identification of novel molecular targets and more effective therapeutics. Cadherin-11, a calcium-dependent cell-cell adhesion molecule and mesenchymal marker, plays a role in both normal tissue development and in cancer cell migration. The functional significance of cadherin-11 in GBM has not been investigated. Here, we show that cadherin-11 is expressed in human GBM tumors and human glioma stem-like cells by immunohistochemical labeling. In addition, we show that cadherin-11 is expressed in human glioma cell lines by immunoblotting. Short hairpin RNA-mediated knockdown of cadherin-11 expression in human glioma cell lines results in decreased migration and growth factor-independent cell survival in vitro. More importantly, knockdown of cadherin-11 inhibits glioma cell survival in heterotopic and orthotopic mouse xenograft models. Together, our results show the functional significance of cadherin-11 expression in GBM and provide evidence for a novel role of cadherin-11 in promoting glioma cell survival in an in vivo environment. Thus, our studies suggest cadherin-11 is a viable molecular target for therapeutic intervention in GBM.

Should I stay or should I go? Shedding of RPTPs in cancer cells switches signals from stabilizing cell-cell adhesion to driving cell migration.

Dissolution of cell-cell adhesive contacts and increased cell-extracellular matrix adhesion are hallmarks of the migratory and invasive phenotype of cancer cells. These changes are facilitated by growth factor binding to receptor protein tyrosine kinases (RTKs). In normal cells, cell-cell adhesion molecules (CAMs), including some receptor protein tyrosine phosphatases (RPTPs), antagonize RTK signaling by promoting adhesion over migration. In cancer, RTK signaling is constitutive due to mutated or amplified RTKs, which leads to growth factor independence, or autonomy. An alternative route for a tumor cell to achieve autonomy is to inactivate cell-cell CAMs such as RPTPs. RPTPs directly mediate cell adhesion and regulate both cadherin-dependent adhesion and signaling. In addition, RPTPs antagonize RTK signaling by dephosphorylating molecules activated following ligand binding. Both RPTPs and cadherins are downregulated in tumor cells by cleavage at the cell surface. This results in shedding of the extracellular, adhesive segment and displacement of the intracellular segment, altering its subcellular localization and access to substrates or binding partners. In this commentary we discuss the signals that are altered following RPTP and cadherin cleavage to promote cell migration. Tumor cells both step on the gas (RTKs) and disconnect the brakes (RPTPs and cadherins) during their invasive and metastatic journey.

Identification of phospholipase C gamma1 as a protein tyrosine phosphatase mu substrate that regulates cell migration.

The receptor protein tyrosine phosphatase PTPµ has a cell-adhesion molecule-like extracellular segment and a catalytically active intracellular segment. This structure gives PTPµ the ability to transduce signals in response to cell-cell adhesion. Full-length PTPµ is down-regulated in glioma cells by proteolysis which is linked to increased migration of these cells in the brain. To gain insight into the substrates PTPµ may be dephosphorylating to suppress glioma cell migration, we used a substrate trapping method to identify PTPµ substrates in tumor cell lines. We identified both PKCδ and PLCγ1 as PTPµ substrates. As PLCγ1 activation is linked to increased invasion of cancer cells, we set out to determine whether PTPµ may be upstream of PLCγ1 in regulating glioma cell migration. We conducted brain slice assays using U87-MG human glioma cells in which PTPµ expression was reduced by shRNA to induce migration. Treatment of the same cells with PTPµ shRNA and a PLCγ1 inhibitor prevented migration of the cells within the brain slice. These data suggest that PLCγ1 is downstream of PTPµ and that dephosphorylation of PLCγ1 is likely to be a major pathway through which PTPµ suppresses glioma cell migration.

Proteolytic cleavage of protein tyrosine phosphatase mu regulates glioblastoma cell migration.

Glioblastoma multiforme (GBM), the most common malignant primary brain tumor, represents a significant disease burden. GBM tumor cells disperse extensively throughout the brain parenchyma, and the need for tumor-specific drug targets and pharmacologic agents to inhibit cell migration and dispersal is great. The receptor protein tyrosine phosphatase mu (PTPmu) is a homophilic cell adhesion molecule. The full-length form of PTPmu is down-regulated in human glioblastoma. In this article, overexpression of full-length PTPmu is shown to suppress migration and survival of glioblastoma cells. Additionally, proteolytic cleavage is shown to be the mechanism of PTPmu down-regulation in glioblastoma cells. Proteolysis of PTPmu generates a series of proteolytic fragments, including a soluble catalytic intracellular domain fragment that translocates to the nucleus. Only proteolyzed PTPmu fragments are detected in human glioblastomas. Short hairpin RNA-mediated down-regulation of PTPmu fragments decreases glioblastoma cell migration and survival. A peptide inhibitor of PTPmu function blocks fragment-induced glioblastoma cell migration, which may prove to be of therapeutic value in GBM treatment. These data suggest that loss of cell surface PTPmu by proteolysis generates catalytically active PTPmu fragments that contribute to migration and survival of glioblastoma cells.

PTPmu suppresses glioma cell migration and dispersal.

The cell-surface receptor protein tyrosine phosphatase mu (PTPmu) is a homophilic cell adhesion molecule expressed in CNS neurons and glia. Glioblastomas (GBMs) are the highest grade of primary brain tumors with astrocytic similarity and are characterized by marked dispersal of tumor cells. PTPmu expression was examined in human GBM, low-grade astrocytoma, and normal brain tissue. These studies revealed a striking loss of PTPmu protein expression in highly dispersive GBMs compared to less dispersive low-grade astrocytomas and normal brain. We hypothesized that PTPmu contributes to contact inhibition of glial cell migration by transducing signals in response to cell adhesion. Therefore, loss of PTPmu may contribute to the extensive dispersal of GBMs. The migration of brain tumor cells was assessed in vitro using a scratch wound assay. Parental U-87 MG cells express PTPmu and exhibited limited migration. However, short-hairpin RNA (shRNA)-mediated knockdown of PTPmu induced a morphological change and increased migration. Next, a brain slice assay replicating the three-dimensional environment of the brain was used. To assess migration, labeled U-87 MG glioma cells were injected into adult rat brain slices, and their movement was followed over time. Parental U-87 MG cells demonstrated limited dispersal in this assay. However, PTPmu shRNA induced migration and dispersal of U-87 MG cells in the brain slice. Finally, in a mouse xenograft model of intracranially injected U-87 MG cells, PTPmu shRNA induced morphological heterogeneity in these xenografts. Together, these data suggest that loss of PTPmu in human GBMs contributes to tumor cell migration and dispersal, implicating loss of PTPmu in glioma progression.

BCCIP associates with the receptor protein tyrosine phosphatase PTPmu.

The receptor protein tyrosine phosphatase PTPmu belongs to a family of adhesion molecules that contain cell-cell adhesion motifs in their extracellular segments and catalytic domains within their intracellular segments. The ability of PTPmu both to mediate adhesion and exhibit enzymatic activity makes PTPmu an excellent candidate to transduce signals in response to cell-cell adhesion. In an effort to identify downstream signaling partners of PTPmu, we performed a modified yeast two-hybrid screen using the first tyrosine phosphatase domain of PTPmu as bait. We isolated an interacting clone encoding BRCA2 and CDKN1A interacting protein (BCCIP) from a HeLa cell library. BCCIP is a p21 and BRCA2 interacting protein that has been shown to play roles in both cell cycle arrest and DNA repair. In this manuscript, we confirm the interaction between BCCIP and PTPmu identified in yeast using in vitro biochemical studies and characterize BCCIP as a PTPmu binding protein. We demonstrate that BCCIP is phosphorylated by the Src tyrosine kinase and dephosphorylated by the PTPmu tyrosine phosphatase in vitro. Furthermore, we show that BCCIP is required for both the permissive and repulsive functions of PTPmu in neurite outgrowth assays, suggesting BCCIP and PTPmu are in a common signal transduction pathway.

The receptor protein-tyrosine phosphatase PTPmu interacts with IQGAP1.

The receptor protein-tyrosine phosphatase PTPmu is a member of the Ig superfamily of cell adhesion molecules. The extracellular domain of PTPmu contains motifs commonly found in cell adhesion molecules. The intracellular domain of PTPmu contains two conserved catalytic domains, only the membrane-proximal domain has catalytic activity. The unique features of PTPmu make it an attractive molecule to transduce signals upon cell-cell contact. PTPmu has been shown to regulate cadherin-mediated cell adhesion, neurite outgrowth, and axon guidance. Protein kinase C is a component of the PTPmu signaling pathway utilized to regulate these events. To aid in the further characterization of PTPmu signaling pathways, we used a series of GST-PTPmu fusion proteins, including catalytically inactive and substrate trapping mutants, to identify PTPmu-interacting proteins. We identified IQGAP1, a known regulator of the Rho GTPases, Cdc42 and Rac1, as a novel PTPmu-interacting protein. We show that this interaction is due to direct binding. In addition, we demonstrate that amino acid residues 765-958 of PTPmu, which include the juxtamembrane domain and 35 residues of the first phosphatase domain, mediate the binding to IQGAP1. Furthermore, we demonstrate that constitutively active Cdc42, and to a lesser extent Rac1, enhances the interaction of PTPmu and IQGAP1. These data indicate PTPmu may regulate Rho-GTPase-dependent functions of IQGAP1 and suggest that IQGAP1 is a component of the PTPmu signaling pathway. In support of this, we show that a peptide that competes IQGAP1 binding to Rho GTPases blocks PTPmu-mediated neurite outgrowth.

Alpha-thrombin-mediated phosphatidylinositol 3-kinase activation through release of Gbetagamma dimers from Galphaq and Galphai2.

Chinese hamster embryonic fibroblasts (IIC9 cells) express the Galpha subunits Galphas, Galphai2, Galphai3, Galphao, Galpha(q/11), and Galpha13. Consistent with reports in other cell types, alpha-thrombin stimulates a subset of the expressed G proteins in IIC9 cells, namely Gi2, G13, and Gq as measured by an in vitro membrane [35S]guanosine 5'-O-(3-thio)triphosphate binding assay. Using specific Galpha peptides, which block coupling of G-protein receptors to selective G proteins, as well as dominant negative xanthine nucleotide-binding Galpha mutants, we show that activation of the phosphatidylinositol 3-kinase/Akt pathway is dependent on Gq and Gi2. To examine the role of the two G proteins, we examined the events upstream of PI 3-kinase. The activation of the PI 3-kinase/Akt pathway by alpha-thrombin in IIC9 cells is blocked by the expression of dominant negative Ras and beta-arrestin1 (Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2000) J. Biol. Chem. 275, 18046-18053, and Goel, R., Phillips-Mason, P. J., Raben, D. M., and Baldassare, J. J. (2002) J. Biol. Chem. 277, 18640-18648), indicating a role for Ras and beta-arrestin1. Interestingly, inhibition of Gi2 and Gq activation blocks Ras activation and beta-arrestin1 membrane translocation, respectively. Furthermore, expression of the Gbetagamma sequestrant, alpha-transducin, inhibits both Ras activation and membrane translocation of beta-arrestin1, suggesting that Gbetagamma dimers from Galphai2 and Galphaq activate different effectors to coordinately regulate the PI 3-kinase/Akt pathway.

alpha-Thrombin activates Akt via a nonreceptor tyrosine kinase in IIC9 cells.

Previous data from our laboratory show that PI 3-kinase is required for alpha-thrombin-stimulated G(1) progression in IIC9 cells. In IIC9 cells, PI 3-kinase acts downstream of Ras to activate Akt, in a pathway parallel to ERK1. Here we show that alpha-thrombin does not transactivate either the EGF receptor or the PDGF receptor as measured by tyrosine phosphorylation, suggesting that activation of PI 3-kinase by alpha-thrombin is not the result of an RTK. Interestingly, both genistein and PP1 block alpha-thrombin-stimulated Akt phosphorylation, suggesting the involvement of a member of the Src family of nonreceptor tyrosine kinases.

alpha-Thrombin induces rapid and sustained Akt phosphorylation by beta-arrestin1-dependent and -independent mechanisms, and only the sustained Akt phosphorylation is essential for G1 phase progression.

In Chinese hamster embryonic fibroblasts (IIC9 cells) alpha-thrombin activates the MAPK(ERK) and phosphatidylinositol 3-OH-kinase (PI 3-kinase)/Akt pathways, and both are essential for progression through the G(1) phase of the cell cycle. We investigated in IIC9 cells, the role of beta-arrestin1 in alpha-thrombin signaling to these pathways. alpha-Thrombin stimulates rapid and sustained PI 3-kinase and Akt activities. Expression of a dominant negative beta-arrestin1 (beta-arrestin1(V53D)) inhibits rapid but not sustained PI 3-kinase and Akt activities. Surprisingly, expression of beta-arrestin1(V53D) does not block activation of the MAPK(ERK) pathway. PI 3-kinase and Akt activities are also inhibited by expression of a beta-arrestin1 mutant, which impairs binding to c-Src (beta-arrestin1(P91G-P121E)), indicating the involvement of c-Src in the rapid stimulation of the PI 3-kinase/Akt pathway. Consistent with these results, PP1, a selective inhibitor of c-Src family kinases, prevents alpha-thrombin-stimulated Akt phosphorylation. Expression of beta- arrestin1(V53D) does not prevent G(1) progression, as its expression has no effect on [(3)H]thymidine incorporation into DNA. In agreement with the ineffectiveness of beta-arrestin1(V53D) to block G(1) progression, cyclin D1 protein amounts and CDK4-cyclin D1 activity is unaffected by expression of beta-arrestin1(V53D). Thus in IIC9 cells, alpha-thrombin activates rapid beta-arrestin1-dependent and sustained beta-arrestin1-independent Akt activity, suggesting that two mechanisms are involved. Furthermore, although blocking the beta-arrestin1-independent PI 3-kinase/Akt pathway prevents G(1) progression, inhibition of the beta-arrestin1-dependent pathway does not, indicating different roles for the rapid and sustained activities.

A novel role for Gq alpha in alpha-thrombin-mediated mitogenic signalling pathways.

alpha-Thrombin activates several G-proteins including members of the Gq, Gi, and G12/13 families, although the physiological importance of these proteins is still not completely understood. We specifically investigated the role of Gq alpha in modulating alpha-thrombin-induced mitogenesis. In Gqa1 cells, a stable cell line expressing reduced amounts of Gq alpha, concentrations of alpha-thrombin (1 NIH unit/ml), which induce cell cycle reentry and progression into S phase in wild-type IIC9 cells, do not stimulate phosphatidylinositol (PI) hydrolysis, the rapid early phase of ERK activity, and transit through G1 into S phase as quantified by cyclin-dependent kinase (CDK)4-cyclin D activity and [3H]thymidine incorporation. Interestingly, high concentrations of alpha-thrombin restore these activities and cell cycle progression into S phase. While, it is well documented that alpha-thrombin-induced sustained ERK activity mediates important responses for transit through G1 into S phase, the importance of the rapid, Gq-dependent phase as a prerequisite for alpha-thrombin-mediated mitogenesis has not been appreciated.