Conditional expression of the dual-specificity phosphatase PYST1/MKP-3 inhibits phosphorylation of cytosolic phospholipase A2 in Chinese hamster ovary cells.
Stimulation of platelet-activating factor (PAF) receptor induces activation of extracellular signal-regulated kinase (ERK) and cytosolic phospholipase A2 (cPLA2) and release of arachidonic acid in Chinese hamster ovary cells. To determine whether the dual-specificity protein phosphatase PYST1/MKP-3 inhibits phosphorylation of cPLA2, we have generated a cell line that conditionally expresses PYST1 under the control of a tetracycline-regulated inducible system. We found that induction of PYST1 suppressed phosphorylation and activation of cPLA2 as well as ERK. Arachidonic acid release was also reduced by about 30%. Pretreatment of cells with an MEK inhibitor, PD98059, had similar effects on PAF-induced cPLA2 phosphorylation and arachidonic acid release. These experiments demonstrate that expression of PYST1 prevents phosphorylation of a cytoplasmic substrate for ERK. Thus, this inducible system may offer a valuable means of investigating physiological roles of ERK in vivo.
Hirabayashi
T
T
Department of Biochemistry and Molecular Biology, University of Tokyo, Japan.
Kume
K
K
Shimizu
T
T
D000818:Animals; D016718:Arachidonic Acid / Q000378:metabolism; D016466:CHO Cells / Q000201:enzymology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D006224:Cricetinae; D003600:Cytosol / Q000201:enzymology*; D054642:Dual Specificity Phosphatase 6; D004789:Enzyme Activation; D006168:Guinea Pigs; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology; D010741:Phospholipases A / Q000037:antagonists & inhibitors* / Q000378:metabolism; D054467:Phospholipases A2; D010766:Phosphorylation; D010980:Platelet Membrane Glycoproteins / Q000096:biosynthesis / Q000235:genetics; D017027:Protein Tyrosine Phosphatases / Q000096:biosynthesis* / Q000378:metabolism / Q000502:physiology; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled
D009097:Multienzyme Complexes; D010980:Platelet Membrane Glycoproteins; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; C039351:platelet activating factor receptor; D016718:Arachidonic Acid; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010741:Phospholipases A; D054467:Phospholipases A2; D054642:Dual Specificity Phosphatase 6; D017027:Protein Tyrosine Phosphatases