Browsing by Author "Moniz, S."
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- Um novo mecanismo molecular para a regulação do transportador de glicose GLUT1Publication . Mendes, A.I.; Matos, P.; Moniz, S.; Jordan, P.One mechanism by which cells regulate the uptake of glucose is the number of glucose transporter proteins (GLUT) present at the plasma membrane. In insulin-responsive cells types, GLUT4 is released from intracellular storage vesicles through inactivation of the Rab GTPase activating protein TBC1D4, also known as AS160. Using protein biochemical techniques we analysed complex formation between TBC1D4 and protein kinase WNK1 in human embryonic kidney (HEK293) cells. We found that WNK1 associates in a protein complex with TBC1D4 and phosphorylates TBC1D4 in vitro. This resulted in increased expression levels of the constitutive glucose transporter GLUT1 at the cell surface. WNK1 was found to increase the binding of TBC1D4 to regulatory 14-3-3 proteins while reducing its interaction with the exocytic small GTPase Rab8A. These effects were dependent on the catalytic activity because expression of a kinase-dead WNK1 mutant had no effect on binding of 14-3-3 and Rab8A, or on surface GLUT1 levels. Together, the data describe a molecular pathway regulating constitutive glucose uptake via GLUT1, the expression level of which is related to several human diseases. This pathway may be important in the cellular response to insulin.
- Protein kinase WNK2 as a tumour suppressor gene in malignant gliomasPublication . Moniz, S.; Martinho, O.; Reis, R.M.; Jordan, P.Malignant glioblastomas are the most common and lethal adult brain tumours, with patients dying within two years from diagnosis. Little is known about the molecular mechanisms underlying the formation and/or development of these tumours, which present a very invasive phenotype within the brain and are genetically heterogeneous and highly resistant to both chemo- and radio-therapies. Recently, the promoter region of the protein kinase WNK2 gene was found to be hypermethylated in 29 of 31 infiltrative gliomas and about 80% of meningiomas. We have previously described that the experimental depletion of WNK2 expression decreases RhoA activity whilst leading to increased Rac1 activity. Because RhoA/Rac1 activities are important for cell migration and glioblastomas are very invasive tumours, we tested the effects of WNK2 on wound-healing assays in glioma cell lines SW1088 and A172. SW1088 cells express endogenous WNK2 and we observed that wound closure was increased upon experimental depletion of endogenous WNK2. In contrast, A172 cells display complete promoter region methylation and WNK2 re-expression was found to decrease migration. Consistently, we observed an increase in Rac1 activity in SW1088 cells upon WNK2 down-regulation, but lower levels of active Rac1 in A172 cells stably expressing WNK2 cDNA when compared with an equivalent cell line stably transfected with the same empty vector. Our studies indicate that loss of WNK2 expression promotes Rac1 activation and may contribute to the highly invasive phenotype that glioblastomas present. We also observed that, in a panel of glioblastoma cell lines, WNK2 promoter methylation correlates with a marked deregulation in Akt, MEK1/2 and ERK1/2 activities, suggesting WNK2 may also be important for tumour cell survival and cell cycle progression.
- Protein kinase WNK2 regulates cell migration in malignant gliomasPublication . Moniz, S.; Martinho, O.; Reis, R.M.; Jordan, P.Malignant glioblastomas are the most common and lethal adult brain tumours, with patients dying within two years from diagnosis. Little is known about the molecular mechanisms underlying the formation and/or development of these tumours, which present a very invasive phenotype within the brain and are genetically heterogeneous and highly resistant to both chemo- and radio-therapies. Recently, the promoter region of the protein kinase WNK2 gene was found to be hypermethylated in 29 of 31 infiltrative gliomas and about 80% of meningiomas. We have previously described that the experimental depletion of WNK2 expression decreases RhoA activity whilst leading to increased Rac1 activity. Because RhoA/Rac1 activities are important for cell migration and glioblastomas are very invasive tumours, we tested the effects of WNK2 on wound-healing assays in glioma cell lines SW1088 and A172. SW1088 cells express endogenous WNK2 and we observed that wound closure was increased upon experimental depletion of endogenous WNK2. In contrast, A172 cells display complete promoter region methylation and WNK2 re-expression was found to decrease migration. Consistently, we observed an increase in Rac1 activity in SW1088 cells upon WNK2 down-regulation, but lower levels of active Rac1 in A172 cells stably expressing WNK2 cDNA when compared with an equivalent cell line stably transfected with the same empty vector. Our studies indicate that loss of WNK2 expression promotes Rac1 activation and may contribute to the highly invasive phenotype that glioblastomas present. We also observed that, in a panel of glioblastoma cell lines, WNK2 promoter methylation correlates with a marked deregulation in Akt, MEK1/2 and ERK1/2 activities, suggesting WNK2 may also be important for tumour cell survival and cell cycle progression.