Fig 2: Protein phosphatase 2A (PP2A) specifically dephosphorylates cytoplasmic ATR at S428 and its action involves direct binding to ATR. (A) A549 cells were treated with siRNAs against three protein phosphatases (PP2Ac, PP4c, and PP5c) followed by UV treatment at 40 J/m2 with a 2 h recovery. Analysis of cytoplasmic extracts reveals that PP2A is required to dephosphorylate cytoplasmic pATR (S428). (B) siRNA knockdown of PP2Ac in A549 cells was followed by UV treatment at 40 J/m2 with a 2 h recovery. pATR (S428) levels are increased in cytoplasm with PP2Ac knockdown while the phosphorylation status of ATR in the nucleus remains unchanged. WB of whole cell extracts shows PP2A siRNA knockdown efficiency in A549 cells (C) HCT 116 ATRflox/– cells were transfected with N-terminal Flag-tagged wild-type (WT) or S428A mutant ATR expression constructs and UV treated at 40 J/m2 with a 2 h recovery. Analysis of the cytoplasmic extracts reveals that PP2A specifically targets the pATR (S428) residue. The pATR (S428) observed in the S428A cells reflects some phosphorylation of the residual endogenous ATR remaining in the ATRflox/– cells (right panel). (D) PLA revealing that in A549 cells there is a direct interaction between ATR and PP2A, especially after UV irradiation at 40 J/m2. A DAPI-staining overlay is used to show location of the nuclei. The bar graph represents a statistical analysis of the PLA images. (E) A549 cells treated with UV at 40 J/m2 were lysed, followed by co-immunoprecipitation of PP2A using anti-ATR antibody. *Stands for p value < 0.01.

Fig 3: Protein phosphatase 2A (PP2A) dephosphorylation of pATR changes ATR to the higher form (ATR-H). (A) UV irradiation of A549 cells at 40 J/m2 increases the formation of ATR-H in the cytoplasm. Also, the ATR-H is not phosphorylated while the ATR-L band is phosphorylated in both the nucleus and the cytoplasm (cytoplasm to nucleus loading ratio = 3:1). The samples were analyzed on a 3–8% gradient gel. (B) A549 cells were pretreated with DMSO (Ctrl) or transfected with PP2A siRNAs, then UV-irradiated at 40 J/m2 (–/+UV) followed by a 2 h recovery. The cytoplasmic fraction was analyzed by electrophoresis through a 3–8% gradient gel and WB to show the form of ATR in the cytoplasm. PP2A depletion reduces the levels of cytoplasmic ATR and the amount of ATR-H formed following UV treatment. (C) WB showing PP2A siRNA knockdown efficiency in A549 cells.

Fig 4: NatB inactivation decreased the level of MAPK and proposed model of EGFR/MAPK signaling regulation by NatB and the N-end rule pathways.(A-B) psid-D1 (A) or psid-D4 (B) clones in 3rd instar fat body, which were marked by GFP expression (pointed by arrowheads), showed decreased MAPK levels (anti-MAPK staining, red). (C) psid-D4 clones in wing disc, marked by GFP expression (pointed by arrowheads), showed decreased MAPK level. (D-E) Ubr4 RNAi clones, marked by GFP expression (pointed by arrowheads), showed increased MAPK level (D). Ubr4 RNAi also restored the level of MAPK in psid-D4 clones (C, E). p35 expression, which did not affect MAPK levels (G), was used to block the death of Ubr4 RNAi cells. (F) The specificity of the anti-MAPK antibody. Reduced MAPK staining was observed in MAPK RNAi clones marked by GFP expression (pointed by arrowheads). (H-I) Ubr1 RNAi (H) or Ubr5 RNAi (I) clones (pointed by arrowheads) did not significantly affect MAPK levels. (J) A proposed model of EGFR/MAPK signaling regulation by NatB and the N-end rule pathways through inversely modulating the levels of positive and negative pathway components is shown. Drk/Grb2 and MAPK, two positive components of the EGFR signaling pathway are targeted for degradation by the Arg/N-end rule pathway and E3 ubiquitin ligase Ubr4 while PP2AC, a negative regulator of EGFR signaling pathway, is targeted for degradation by the Ac/N-end rule pathway E3 ubiquitin ligase Cnot4. The levels of acetylation of these proteins are controlled by NatB activity and the key rate-limiting substrate Ac-CoA. High NatB activity and high Ac-CoA level will increase the levels of Nt-acetylated Grb2/Drk, MAPK, and PP2AC, leading to high EGFR signaling due to elevated levels of Grb2/Drk and MAPK (positive components of the pathway) and reduced levels of PP2AC (negative component of the pathway). In contrast, low NatB activity and low Ac-CoA levels will reduce the levels of Nt-acetylated Grb2/Drk, MAPK, and PP2AC, leading to reduced EGFR signaling due to decreased levels of Grb2/Drk and MAPK (positive components of the pathway) and increased levels of PP2AC (negative component of the pathway). (K) psid mutation inhibited Ey-FLP induced MARCM clones of activated EGFR expression (labelled with GFP) in scrib mutant clones. Yellow and white arrows point to tumor growth in the head region and the male gonads, respectively. (L) Expression of WT Psid or the Psid-SA mutant but not the NAA20-binding defective Psid-SD mutant restored tumor growth, suggesting the NatB activity is required for activated EGFR-induced tumor growth. (M-N’) psid mutation significantly inhibited activated EGFR-induced cell proliferation in male gonads. scrib mutant clones with activated EGFR expression (pointed by arrowhead) were highly proliferative with high level of BrdU incorporation (M-M’, red). In contrast, mutation of psid significantly inhibited BrdU incorporation in EGFR scrib cells (N-N’, arrowhead pointed GFP positive cells). Arrows in N-N’ point to the normal proliferating cells in the anterior pole.

Fig 5: Protein phosphatase 2A (PP2A) dephosphorylates cytoplasmic pATR (S428) in a UV dose- and recovery time-dependent manner and is independent of ATRIP. (A) Cytoplasmic pATR (S428) dephosphorylation by PP2A is dependent on post-irradiation recovery time in A549 cells. The WB shows that the level of ATR phosphorylation at S428 depends on the recovery time following UV irradiation at 40 J/m2. PP2A inhibitor treatment (PP2Ai) abolishes this dephosphorylation at all recovery times. (B) Cytoplasmic pATR (S428) dephosphorylation by PP2A is UV dose dependent and is attenuated with PP2A inhibitor treatment of A549 cells. Control A549 cells show a reduction in the level of pATR (S428) that is dependent on the dose of UV irradiation used, but retain their level of pATR (S428). (C) A549 cells, depleted of ATRIP by siRNA knockdown, were divided into two groups with one treated with PP2A inhibitor and the other left as a control. WB assay shows that this ATRIP knockdown does not affect PP2A’s dephosphorylation of pATR (S428) in the cytoplasm of A549 cells. The right panel shows the efficiency of the ATRIP knockdown.