Abstract
Phosphoinositide-specific inositol polyphosphate 5- phosphatase IV has the affinity for PI(3,4,5)P(3) (K(m) = 0.65 microM) that is approximately 10-fold greater than the other inositol polyphosphate 5-phosphatases, which use this substrate including SHIP, OCRL, and 5ptase II, suggesting that it may be important in controlling intracellular levels of this metabolite. We created cell lines stably expressing the enzyme to study its effect on cell function. We found that overexpression of 5ptase IV in 293 cells caused the rapid depletion of both PI(4,5)P(2) and PI(3,4,5)P(3) in cells with corresponding increases in the products, PI(4)P and PI(3,4)P(2), changing the balance of two phosphoinositol products of phosphoinositide 3-kinase, PI(3,4)P(2) and PI(3,4,5)P(3), in the cell. One of the targets of these phosphoinositides is the serine/threonine kinase Akt, which plays an important role in the control of apoptosis. We were able to address the relative roles of PI(3,4)P(2) and PI(3,4,5)P(3) in the activation of Akt by selective depletion of these phosphoinositides in cells stably transfected with 5ptase IV and inositol polyphosphate 4-phosphatase (4ptase I). In cells transfected with 4ptase I, the level of PI(3,4)P(2) was reduced, and PI(3,4,5)P(3) was increased. Expression of the two enzymes had the opposite effect on the phosphorylation of Akt in response to stimulation with growth factors or heat shock. Akt phosphorylation was inhibited in cells expressing 5ptase IV but increased in 4ptase I cells and correlated with the intracellular level of PI(3,4,5)P(3) and not that of PI(3,4)P(2). The inhibition of Akt phosphorylation in cells expressing 5ptase IV makes them highly susceptible to FAS-induced apoptosis, whereas overexpressing of the 4ptase I protects cells from apoptosis. Our results place 5ptase IV as a relevant biological regulator of PI3K/Akt pathway in cells.
Highlights
Phosphatidylinositol lipids, referred to as phosphoinositides, are involved in the regulation of an array of cellular processes, including cell growth, differentiation, cell motility, and apoptosis
Phosphoinositide-specific inositol polyphosphate 5phosphatase IV has the affinity for PI[3,4,5]P3 (Km ؍0.65 M) that is approximately 10-fold greater than the other inositol polyphosphate 5-phosphatases, which use this substrate including SHIP, OCRL, and 5ptase II, suggesting that it may be important in controlling intracellular levels of this metabolite
We found that overexpression of 5ptase IV in 293 cells caused the rapid depletion of both PI[4,5]P2 and PI[3,4,5]P3 in cells with corresponding increases in the products, PI[4]P and PI[3,4]P2, changing the balance of two phosphoinositol products of phosphoinositide 3-kinase, PI[3,4]P2 and PI[3,4,5]P3, in the cell
Summary
Generation of Cell Lines Stably Expressing Inositol Polyphosphate Phosphatases—The T-Rex tetracycline-regulated system (Invitrogen) was used to create cell lines stably expressing inositol phosphatases. cDNA encoding Myc-tagged full-length 5ptase IV (GenBankTM AF187891) was subcloned into the BamHI site of pcDNA 4/TO vector (Invitrogen). 5ptase, inositol polyphosphate 5-phospatase; 4ptase, inositol polyphosphate 4-phospatase; PI[4]P, phosphatidylinositol 4-phosphate; PI[3,4]P2, phosphatidylinositol 3,4-bisphosphate; PI[4,5]P2, phosphatidylinositol 4,5-bisphosphate, PI[3,4,5]P3, phosphatidylinositol 3,4,5trisphosphate; FBS, fetal bovine serum; LDH, lactate dehydrogenase. An addition of 100 ng of tetracycline/ml to the growth medium induced the expression of the inositol phosphatase proteins. Protein expression was induced by the addition of 100 ng of tetracycline/ml in Dulbecco’s modified Eagle’s medium supplemented with 5% FBS. Induction of Apoptosis—0.8 ϫ 106 cells were plated/well in a 12-well plate, and protein expression was induced by overnight incubation in a medium containing 100 ng of tetracycline/ml. The extend of apoptosis was measured as a -fold increase of LDH activity in the sample compared with LDH activity measured in the media of untreated vector cells
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
