has been shown to be a highly efficient inierfacial AKT phosphoryliion in a seco

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has been shown to be a highly efficient inierfacial AKT phosphoryliion in a second PTEN deficient We esplored the efflect of overespression o phosphatase (2/). We also found that coexpres glioa c line(ig S10) In plaic-darived son of P-REX2a, but not ?DHPH. decreased grov th factor (PDGFH t mulated U87-MG cells he phosphatase activity of PTEN immunopre (fig, SI2) P-REX2a on endogenous wild-type PTEN in an imot la dhann mummu y cell line. MCF10A. cipitated fiom these cells dig. SS) Using a alite reduced unes of plapuntation Of DHPH etteted ???esed sof phoplo- point mutant modeled after P-REXI, we fur- T308AKT S473AKT, and glycogem synthetase tylation of Se AKT and enhanced proleration her showed that PTEN phosphatase nhhition kinac 3 ? GSKSB) P-KEX2acoespresan with in tssuc culture (Fig. 2, D and Ei The presence was indapcndant of the GEF activity of P-REX2a PTEN orad lavels of phosphoryated AKT and of gowth factors was roquirod to obsrve this diference in proliferation P-REX2a may nit be Expressacn of PTEN in PTENdeficient U87- tigaled P-REX2a also was able to recue PTEN suficient ? mpur,mwth far mdependent MG cells decrcasod the amount of phospborylatod supssion of insulin signaling but was incfoctiual prolufcration, because efects of PTEN inhibition AKTIpAKT) (Fig. 2C and figs. S10 to S12). fr tosc cclls?mulaod with EGF·?Sl31 would stil roquire PlP3 gonantion to allow for (ig 59) (22 GSK3ßto baseline Aneg der agants ? es, Ovemexpression of P-REXZa had mo elfect on Thoughoul these experimens, we ohserved thal PI3K activation AAKT alone, but when expressed with PTEN, ? wildeype P-REX2a expression ucreased levels of simultaneously overexpressed PTE0Fig. ? Analysis of P-REX2a ERNA in a cobeet of presang brea, tum os showed a s- of AKT at both Scr") on both the titlg S10) and ?alytic actity of have shown that PTEN stability nd Kity are (P-0014, r test) (FIg 3A). Expreson of either P-REXZa or the PI3Ka E54SK (Gu replaced Fig. 4. Diminished phosA phorylation of AKT and proliferation after deple- tion of P REX2a. Phospho ylation of AKT (A) and proliferation 1B) after de- pletion of P-REXZa in PTEN wild-type MCFT cels or PTEN-nul BTS49 celb. 00595, absorbance at 595 nm. (CD Effect of AKT P-REXZa depletion in MCF10A cells on pAKT and pGsk3 and amounts of the p21 and p27 cell cycle inhibitors. D)Xgal staining of indikated cel ines Phase-contrast im- MCF7 ages displayed at 40 magnification E MCF20A proliferation after P-REX 2 1MCFIOA BT549 264 VOL 325 SCIENCE www.sciencemsg org

Explanation / Answer

Fig. A: Phosphorylation of AKT and proliferation

Fig. B: After depletion of PREX2a in PTEN wild type MCF7 or PTEN -null BT549 cells

Fig.C: Effect of PREX2a depletion in MCF110A cells on pAKT and pGSK3 and amounts of the p21 and p27 cell ccycle inhibitors

Fig:D : X-gal staining indicated cell lines with phase contrast images.

Fig.E: MCF10 A proliferation after PREX2a depletion

the effects of depletionof PREX2a on the P13K pathwayand cell proliferationare dependent on the presence of PTEN.Depletion of P-REX2a in MCF10 A cells decresed amount of pAKT and pGSK3 and reduced proliferation rate. Staing of these cells with X-gal increse in blue colur cells in both shRNA kockdown cell line compared to control cells

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