Cdks regulate GATA2 stability. (A) Density plot analysis of 293T cells transfected with GFP-GATA2 expression plasmid after incubation with roscovitine or MG132 for 12 hours. Numbers indicate percentages of cells in 6 regions outlined with thin lines. (B, left panel) decrease of DNA content (X geometric mean channel) of GFP++ (channel > 103) cells. (B, right panel) Change of fluorescence intensity (Y geometric mean channel) of cells containing 2N DNA with lower fluorescence expression (in the region outlined with bold rectangles in A) (mean ± standard deviation, *P < .01, n = 6). (C-G) Interaction and phosphorylation of S/T0P+1 motifs of GATA2 with Cdk/cyclin systems. Total cell lysates of 293T cells, expressing FlagΔNTGATA2 (C) or Flag-GATA2 (D-E) were immunoprecipitated (IP) with anti-Flag antibody, then immunoblotted (IB) with antiphosphoS0P+1 (pSP), antiphosphoT0P+1 (pTP), anti–cyclin A2, anti-Cdk2, and anti-Cdk4 antibodies. (D) In vitro phosphorylation analysis of immunoprecipitated Flag-GATA2 by recombinant Cdk/cyclin proteins. (E) Effect of roscovitine or SB203580 treatment for 12 hours on phosphorylation of GATA2. (F-G) Immunoprecipitation and nickel resin purification of cell lysates expressing Flag-GATA2 and His–cyclin A2. (G) Induction of GATA2 phosphorylation with cotransfected His–cyclin A2.
