Investigation of molecular mechanisms of LKB1 function in Dictyostelium development and stress responses and the function of superoxide dismutase C (SodC) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium . During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC− cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC− cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC− cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC − cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.

Novel insights into the mechanisms of regulation of tyrosine kinase receptors by Ras Interference 1

Receptor-tyrosine kinases (RTKs) are membrane bound receptors characterized by their intrinsic kinase activity. RTK activities play an essential role in several human diseases, including cancer, diabetes and neurodegenerative diseases. RTK activities have been regulated by the expression or silencing of several genes as well as by the utilization of small molecules. Ras Interference 1 (Rin1) is a multifunctional protein that becomes associated with activated RTKs upon ligand stimulation. Rin1 plays a key role in receptor internalization and in signal transduction via activation of Rab5 and association with active form of Ras. This study has two main objectives: (1) It determines the role of Rin1 in the regulation of several RTKs focusing on insulin receptor. This was accomplished by studying the Rin1-insulin receptor interaction using a variety of biochemical and morphological assays. This study shows a novel interaction between the insulin receptor and Rin1 through the Vps9 domain. Two more RTKs (epidermal growth factor receptor and nerve growth factor receptor) also interacted with the SH2 domain of Rin1. The effect of the Rin1-RTK interaction on the activation of both Rab5 and Ras was also studied during receptor internalization and intracellular signaling. Finally, the role of Rin1 was examined in two differentiation processes (adipogenesis and neurogenesis). Rin1 showed a strong inhibitory effect on 3T3-L1 preadipocyte differentiation but it seems to show a modest effect in PC12 neurite outgrowth. These data indicate a selective function and specific interaction of Rin1 toward RTKs. (2) It examines the role of the small molecule Dehydroleucodine (DhL) on several key signaling molecules during adipogenesis. This was accomplished by studying the differentiation of 3T3-L1 preadipocytes exposed to different concentrations of DhL in different days of the adipocyte formation process. The results indicate that DhL selectively blocked adipocyte formation, as well as the expression of PPARγ, and C/EBP&agr;. However, DhL treatment did not affect Rin1 or Rab5 expression and their activities. Taken together, the data indicate a potential molecular mechanism by which proteins or small molecules regulate selective and specific RTK intracellular membrane trafficking and signaling during cell growth and differentiation in normal and pathological conditions.

Investigation of molecular mechanisms of lkb1 function in dictyostelium development and stress responses and the function of superoxide dismutase c (sodc) in chemotaxis

The serine/threonine kinase LKB1 is a regulator of critical events including development and stress responses in metazoans. The current study was undertaken to determine the function of LKB1 in Dictyostelium. During multicellular development and in response to stress insult, an apparent increase in the DdLKB1 kinase activity was observed. Depletion of DdLKB1 with a knockdown construct led to aberrant development; a severe reduction in prespore cell differentiation and a precocious induction of prestalk cells, which were reminiscent of cells lacking GSK3, a well known cell-fate switch. Furthermore, DdLKB1 depleted cells displayed lower GSK3 activity than wild type cells in response to cAMP stimulation during development and failed to activate AMPK, a well known LKB1 target in mammals, in response to cAMP and stress insults. These results suggest that DdLKB1 positively regulates both GSK3 and AMPK during Dictyostelium development, and DdLKB1 is necessary for AMPK activation during stress response regulation. No apparent GSK3 activation was observed in response to stress insults. Spatial and temporal regulation of phosphatidylinositol-(3,4,5)-triphosphate (PIP3) along the membrane of polarized cells is important for efficient chemotaxis. A REMI screen for PIP3 suppressors in the absence of stimulation led to the identification of SodC as PIP3 regulator. Consistent with their higher PIP3 levels, sodC- cells showed defects in chemotaxis and exhibited higher intra-cellular superoxide levels. Protein localization studies along with observations from GPI specific PI-PLC treatment of wild-type cells suggested that SodC is a GPI anchored outer-membrane protein. SodC showed superoxide dismutase activity in vitro, and motility defects of sodC- cells can be rescued by expressing the intact SodC but not by the mutant SodC, which has point mutations that affect its dismutase function. Treatment of sodC- cells with LY294002, a pharmacological inhibitor of PI3K, partially rescued the polarization and chemoattractant sensing defects but not motility defects. Consistent with increased intracellular superoxide levels, sodC- cells also exhibited higher basal Ras activity, an upstream regulator of PI3K, which can be suppressed by a cell permeable superoxide scavenger, XTT, indicating that SodC is important in regulation of intracellular superoxide levels thereby regulating the Ras activity and PIP3 levels at the membrane.