Inhibition of anchorage-independent cell growth, adhesion, and cyclin D1 gene expression by a dominant negative mutant of a tyrosine phosphatase

PTP-S4/TC48 protein tyrosine phosphatase is localized in the nuclear and cytoplasmic membranes. To investigate the role of PTP-S4 in cell growth, adhesion, and transformation, normal and a catalytically inactive mutant form of this phosphatase were expressed in polyoma virus-transformed F111 fibroblast cell line, PyF. Expression of mutant PTP-S4 in PyF cells resulted in strong inhibition of anchorage-independent growth in soft agar but had no significant effect on growth in liquid culture. Tumor formation in nude mice was also reduced by mutant PTP-S4. Expression of normal PTP-S4 in PyF cells significantly increased anchorage-independent cell growth and tumor formation in nude mice. Overexpression of catalytically inactive mutant of PTP-S2/TC45 (a splice variant of PTP-S4 that is nuclear) did not inhibit anchorage-independent growth of PyF cells. Mutant PTP-S4-expressing cells were inhibited in adhesion and spreading on tissue culture plates compared to control cells. Expression of mutant PTP-S4 in PyF cells reduced the levels of cyclin D1 and cyclin A mRNA, whereas cyclin D2 mRNA level was not affected significantly. Expression of antisense cyclin D1 strongly inhibited anchorage-independent growth. Inhibition of anchorage-independent growth by mutant PTP-S4 was overcome to a large extent by coexpression of cyclin D1. These results suggest that mutant PTP-S4 inhibits anchorage-independent growth and adhesion of polyoma virus-transformed cells by interfering with the normal function of PTP-S4 upstream of cyclin D1 gene expression.     

PTEN,a widely known negative regulator of insulin/PI3K signaling,positively regulates neuronal insulin resistance

Lipid and protein tyrosine phosphatase, phosphatase and tension homologue (PTEN), is a widely known negative regulator of insulin/phosphoinositide 3-kinase signaling. Down-regulation of PTEN is thus widely documented to ameliorate insulin resistance in peripheral tissues such as skeletal muscle and adipose. However, not much is known about its exact role in neuronal insulin signaling and insulin resistance. Moreover, alterations of PTEN in neuronal systems have led to discovery of several unexpected outcomes, including in the neurodegenerative disorder Alzheimer''s disease (AD), which is increasingly being recognized as a brain-specific form of diabetes. In addition, contrary to expectations, its neuron-specific deletion in mice resulted in development of diet-sensitive obesity. The present study shows that PTEN, paradoxically, positively regulates neuronal insulin signaling and glucose uptake. Its down-regulation exacerbates neuronal insulin resistance. The positive role of PTEN in neuronal insulin signaling is likely due to its protein phosphatase actions, which prevents the activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), the kinases critically involved in neuronal energy impairment and neurodegeneration. Results suggest that PTEN acting through FAK, the direct protein substrate of PTEN, prevents ERK activation. Our findings provide an explanation for unexpected outcomes reported earlier with PTEN alterations in neuronal systems and also suggest a novel molecular pathway linking neuronal insulin resistance and AD, the two pathophysiological states demonstrated to be closely linked.     

amiA is a negative regulator of acetamidase expression in Mycobacterium smegmatis

Background  

The acetamidase of Mycobacterium smegmatis is a highly inducible enzyme. Expression of this enzyme is increased 100-fold when the substrate acetamide is present. The acetamidase gene is found immediately downstream of three open reading frames. Two of these are proposed to be involved in regulation.     

PTEN, a negative regulator of PI3 kinase signalling, alters tau phosphorylation in cells by mechanisms independent of GSK-3

Deregulation of PTEN/Akt signalling has been recently implicated in the pathogenesis of Alzheimer's disease (AD), but the effects on the molecular processes underlying AD pathology have not yet been fully described. Here we report that overexpression of PTEN reduces tau phosphorylation in CHO cells. This effect was abrogated by mutant PTEN constructs with either a catalytically inactive point mutation (C124S) or with only inactive lipid phosphatase activity (G129E), suggesting an indirect, lipid phosphatase-dependent process. The predominant effects of PTEN on tau appeared to be mediated by reducing ERK1/2 activity, but were independent of Akt, GSK-3, JNK and the tau phosphatases PP1 and PP2A. Our studies provide evidence for an effect of PTEN on the phosphorylation of tau in AD pathogenesis, and provide some insight into the mechanisms through which deregulation of PTEN may contribute towards the progression of tauopathy.     

Phospholipase D is a negative regulator of proline biosynthesis in Arabidopsis thaliana

Accumulation of proline has been observed in a large number of plant species in response to drought and salt stresses, suggesting a key role of this amino acid in plant stress adaptation. Upstream components of the proline biosynthesis signal transduction pathways are still poorly defined. We provide experimental evidence that phospholipase D (PLD) is involved in the regulation of proline metabolism in Arabidopsis thaliana. The application of primary butyl alcohols, which divert part of PLD-derived phosphatidic acid by transphosphatidylation, stimulated proline biosynthesis even without hyperosmotic constraints. Moreover, application of primary butyl alcohols enhanced the proline responsiveness of seedlings to mild hyperosmotic stress. These data indicate that some PLDs are negative regulators of proline biosynthesis and that plants present a higher proline responsiveness to hyperosmotic stress when this regulator is abolished. We clearly demonstrate that PLD signaling for proline biosynthesis is similar to RD29A gene expression and different from the abscisic acid-dependent RAB18 gene expression. Our data reveal that PLDs play positive and negative roles in hyperosmotic stress signal transduction in plants, contributing to a precise regulation of ion homeostasis and plant salt tolerance.     

Increased cyclin E expression may obviate the role of cyclin D1 during brain development in cyclin D1 knockout mice

Cyclins D and E play critical roles during the G1 phase of mammalian cell division. Cyclin D1 expression is high and expected to play an important role during mouse brain development. However, in the present study, we found no difference in CNS morphology between cyclin D1 knockout (KO) and control wild-type mice at the ages of 1, 4 and 12 months. Analysis of protein expression in embryonic brains revealed that cyclin E is obviously increased in cyclin D1 KO mice at 13.5 days post coitum. At the same age a high level of cyclin D1 expression is detected in the embryonic brain of wild-type mice. The data indicate that enhanced cyclin E protein expression in cyclin D1 KO mice may obviate the role of cyclin D1 and contribute to the normal brain development of cyclin D1 KO mice.     

Disruption of cyclin D3 blocks proliferation of normal B-1a cells, but loss of cyclin D3 is compensated by cyclin D2 in cyclin D3-deficient mice

Peritoneal B-1a cells differ from splenic B-2 cells in the molecular mechanisms that control G(0)-S progression. In contrast to B-2 cells, cyclin D2 is up-regulated in a rapid and transient manner in phorbol ester (PMA)-stimulated B-1a cells, whereas cyclin D3 does not accumulate until late G(1) phase. This nonoverlapping expression of cyclins D2 and D3 suggests distinct functions for these proteins in B-1a cells. To investigate the contribution of cyclin D3 in the proliferation of B-1a cells, we transduced p16(INK4a) peptidyl mimetics (TAT-p16) into B-1a cells before cyclin D3 induction to specifically block cyclin D3-cyclin-dependent kinase 4/6 assembly. TAT-p16 inhibited DNA synthesis in B-1a cells stimulated by PMA, CD40L, or LPS as well as endogenous pRb phosphorylation by cyclin D-cyclin-dependent kinase 4/6. Unexpectedly, however, cyclin D3-deficient B-1a cells proliferated in a manner similar to wild-type B-1a cells following PMA or LPS stimulation. This was due, at least in part, to the compensatory sustained accumulation of cyclin D2 throughout G(0)-S progression. Taken together, experiments in which cyclin D3 was inhibited in real time demonstrate the key role this cyclin plays in normal B-1a cell mitogenesis, whereas experiments with cyclin D3-deficient B-1a cells show that cyclin D2 can compensate for cyclin D3 loss in mutant mice.     

Type 1 phosphatase,a negative regulator of cardiac function

Increases in type 1 phosphatase (PP1) activity have been observed in end stage human heart failure, but the role of this enzyme in cardiac function is unknown. To elucidate the functional significance of increased PP1 activity, we generated models with (i) overexpression of the catalytic subunit of PP1 in murine hearts and (ii) ablation of the PP1-specific inhibitor. Overexpression of PP1 (threefold) was associated with depressed cardiac function, dilated cardiomyopathy, and premature mortality, consistent with heart failure. Ablation of the inhibitor was associated with moderate increases in PP1 activity (23%) and impaired beta-adrenergic contractile responses. Extension of these findings to human heart failure indicated that the increased PP1 activity may be partially due to dephosphorylation or inactivation of its inhibitor. Indeed, expression of a constitutively active inhibitor was associated with rescue of beta-adrenergic responsiveness in failing human myocytes. Thus, PP1 is an important regulator of cardiac function, and inhibition of its activity may represent a novel therapeutic target in heart failure.     

Susi, a negative regulator of Drosophila PI3-kinase

The Phosphatidylinositol-3 kinase/Protein Kinase B (PI3K/PKB) signaling pathway controls growth, metabolism, and lifespan in animals, and deregulation of its activity is associated with diabetes and cancer in humans. Here, we describe Susi, a coiled-coil domain protein that acts as a negative regulator of insulin signaling in Drosophila. Whereas loss of Susi function increases body size, overexpression of Susi reduces growth. We provide genetic evidence that Susi negatively regulates dPI3K activity. Susi directly binds to dP60, the regulatory subunit of dPI3K. Since Susi has no overt similarity to known inhibitors of PI3K/PKB signaling, it defines a novel mechanism by which this signaling cascade is kept in check. The fact that Susi is expressed in a circadian rhythm, with highest levels during the night, suggests that Susi attenuates insulin signaling during the fasting period.