Figure 4. Akt phosphorylates PACS-2 Ser₄₃₇ in vitro and vivo.

(A) Diagram of PACS-2 showing the N-terminal region (NTR), cargo-binding region (FBR), which contains Ser₄₃, the middle region (MR), which contains Ser₂₄₄, Ser₄₃₅ and Ser₄₃₇, and the C-terminal region (CTR). (B) The indicated GST-fusion proteins were incubated with [γ-³²P]-ATP and constitutively active HA-Akt (WT) or kinase-dead HA-Akt (KD). Con, immunoisolation from mock-infected cells. Left, autoradiography; Right, input. (C) The indicated GST-fusion proteins were incubated with [γ-³²P]-ATP and immunoisolated HA-Akt as described in panel B. Top, autoradiography; Bottom, input. (D) Peptides from in-gel limited trypsin digestion of HA-tagged PACS-2 were evaluated for candidate phosphopeptide by mass spectrometry. A phosphopeptide at [M+2H]=450.7, corresponding to the PACS-2 tryptic peptide RSTpS₄₃₇LKER with one phosphorylation, was identified by nanoLC-MS/MS. (E) Left; Brain cytosol from WT and PACS-2^-/- mice was incubated with affinity purified anti-PACS-2 (Ab 832) and immunoprecipitated proteins were analyzed by western blot using anti-PACS-2 (Ab 193) or mAb 81 to detect pSer₄₃₇. Right; Liver and small intestine cytosol from WT mice were analyzed as described above. (F) Lysates from WT and Akt1^-/- MEFs were analyzed as described in panel E to detect pSer₄₃₇. (G) HCT116 cells were harvested in proliferative phase (untreated), serum-starved for 16 hr (starved), starved and refed with 20% serum for 3 hr (control), or starved and pre-treated with 1 μM PI-103, 20 nM RAD011, 1 μM BKM-120 or 20 nM BEZ-235 for one hr prior to refeeding with 20% serum for 3 hr. Cells were analyzed by western blot to detect total PACS-2 and PACS-2 pSer₄₃₇. The pSer₄₃₇:total PACS-2 ratio was calculated and normalized to the untreated sample. Data are represented as mean +/- SD.