A Novel Interaction between hScrib and PP1γ Downregulates ERK Signaling and Suppresses Oncogene-Induced Cell Transformation

<div><p>Previous studies have shown that the cell polarity regulator hScrib interacts with, and consequently controls, the ERK signaling pathway. This interaction occurs through two well-conserved Kinase Interacting Motifs, which allow hScrib to bind ERK1 directly, resulting in a reduction in the levels of phospho-ERK. This suggests that hScrib might recruit a phosphatase to regulate this signaling pathway. Using a proteomic approach we now show that Protein Phosphatase 1γ (PP1γ) is a major interacting partner of hScrib. This interaction is direct and occurs through a conserved PP1γ interaction motif on the hScrib protein, and this interaction appears to be required for hScrib's ability to downregulate ERK phosphorylation. In addition, hScrib also controls the pattern of PP1γ localization, where loss of hScrib enhances the nuclear translocation of PP1γ. Furthermore, we also show that the ability of hScrib to interact with PP1γ is important for the ability of hScrib to suppress oncogene-induced transformation of primary rodent cells. Taken together, these results demonstrate that hScrib acts as a scaffold to integrate the control of the PP1γ and ERK signaling pathways and explains how disruption of hScrib localisation can contribute towards the development of human malignancy.</p> </div

hScrib contains a consensus PP1-binding motif.

<p>A) The schematic shows the arrangement of the functional domains on the hScrib protein, highlighting the LRR, LAPSD and PDZ domains. The putative PP1-binding site, the RVXF (the consensus sequence is K/R/H/N/S V/I/L X F/W/Y) motif is also shown, where X is any amino acid. The hScrib mutant in which the PP1-binding site KLDY was mutated to KADA in order to disrupt the interaction with PP1 is shown. A comparison sequence alignment of the region of hScrib containing the PP1-binding motif indicating its absence in Drosophila also shown. B) In vitro translated and radiolabeled PP1γ was incubated with purified full length GST-hScrib fusion protein (FL), GST-hScrib PDZ1-4 (P1-4), GST-hScrib CT (CT), GST-hScrib L1266Y1268→AA (KADA) and GST alone as a control. After extensive washing the bound PP1γ was ascertained by SDS PAGE and autoradiography. The upper panel shows the autoradiograph, with the input of PP1γ also shown for comparison. The lower panel shows the Coomassie stain of the gel showing the levels of GST fusion protein loading, with the arrows indicating the relevant full length fusion proteins.</p

Interaction between hScrib and PP1γ in vivo.

<p>A) Results from the mass spectroscopy analysis of hScrib containing immunoprecipitates identified 6 peptides (indicated) corresponding to PP1γ. B) In vitro translated PP1γ (upper panels) and PP2A subunit A (lower panels) were incubated for 1 hour at 4°C with purified GST-hScribP1-C or GST alone immobilized on Glutathione agarose. After extensive washing, the bound proteins were analysed by SDS–PAGE and autoradiography which are shown in each of the upper panels. The gels were rehydrated and stained with Coomassie to show equal levels of GST loading in the respective lower panels. C) Endogenous PP1γ was immunoprecipitated from HaCaT (upper panels) and HEK 293 cells (lower panels), with pre-immune antibody used as control. The immunoprecipitated proteins were then analysed by western blotting using anti-hScrib and anti-PP1γ antibodies.</p

PP1γ is required for hScrib-induced de-phosphorylation of ERK.

<p>A) HEK 293 cells were transfected with PP1γ si RNA or si Luc RNA as control (CTL) and after 24 hours were then transfected with a plasmid expressing HA-tagged hScrib. After a further 24 hours the cells were extracted and levels of phospho and total ERK determined by western blot analysis. The upper three panels shows the changes in the ERK profiles when cells were transfected with siRNA PP1γ alone, whilst the lower set of five panels show the effects in the presence of ectopically expressed hScrib. B) HEK 293 cells were transfected with HA-tagged wild type hScrib, or the S1445D and KADA mutants. Total cell extracts were then made after 48 hours and the hScrib, phospho-ERK and total ERK were detected by western blotting.</p

hScrib suppresses HPV-16 E7 and EJ-ras induced transformation in cooperation with PP1γ in a RVxF motif-dependent manner.

<p>BRK cells were transfected with EJ-ras alone, HPV-16 E7 plus EJ-ras, HPV-16 E7 plus EJ-ras and wild type hScrib, HPV-16 E7 plus EJ-ras and PP1γ, and HPV-16 E7 plus EJ-ras and wild type hScrib with PP1γ, and HPV-16 E7 plus EJ-ras and PP1γ plus the KADA non-PP1γ binding mutant of hScrib. After three weeks the dishes were fixed and stained and the colonies counted. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0053752#s3" target="_blank">Results</a> represent the mean number of colonies from 3 independent assays and standard deviations are shown.</p

Loss of hScrib results in enhanced nuclear accumulation of both PP1γ and pERK.

<p>Control and shScrib HaCaT cells were stained for hScrib, phospho-ERK and PP1γ as indicated.</p

hScrib regulates PP1γ nuclear localization.

<p>A) Immunofluorescence analysis of hScrib and PP1γ expression in sh-Luc control HaCaT cells and sh-hScrib knockdown cells. The cells were grown on coverslips and then fixed and double-stained with the anti-hScrib antibody and the anti-PP1γ antibody. Note the significant increase in the levels of nuclear PP1γ in the absence of hScrib expression. B) Z-reconstruction (x-z direction) of a z-stack (15 planes, z-distance 0.2 µm), showing sh-hScrib knockdown cells have enhanced PP1γ localisation into the nucleus. C) HEK 293 cells were transfected with hScrib siRNA and si Luc RNA as control. Cells were either extracted in SDS PAGE sample buffer (Total lysate) or were fractionated into cytoplasmic (F1), membrane (F2) and nuclear (F3) pools (the example shows the integrity of a typical extraction procedure) and then PP1γ was detected by western blotting. p84 was used as a loading control for the nuclear fraction, cadherin was used as a loading control for the membrane fraction and α-tubulin was used as the loading control for the cytoplasmic fraction and total cell extracts. Note the relative increase in nuclear PP1γ following hScrib knockdown but no overall change in total PP1γ levels.</p

hScrib is a substrate of PP1γ.

<p>A) Purified GST-hScrib fusion protein was in vitro phosphorylated with purified PKA or ERK1 as described previously (19) and then incubated with PP1γ for 20 mins at 30°C. Bound PP1γwas detected by western blotting with anti PP1γ antibody. The lower panel shows the ponceau stain of the nitrocellulose, and the upper right panel shows the quantitations from three independent experiments. Note that hScrib phosphorylated by PKA exhibits increased association with PP1γ. B) Purified PP1γ was incubated with purified full length wild type GST-hScrib fusion protein (P1-C), the mutants S1445A, S1445D or GST alone as a control. After extensive washing the bound PP1γ was ascertained by western blotting. The upper panel shows the result of the western blot, with the 20% input of PP1γ also shown for comparison. The lower panel shows the ponceau stain of the nitrocellulose. The histogram shows the quantitation from three independent experiments. C) Purified GST-hScrib wild type and PKA phospho-site mutants of hScrib were in vitro phosphorylated with purified PKA in the presence of radiolabeled ATP as described previously (19) and incubated with PP1γ for 20 mins at 30°C. The remaining level of phosphorylated hScrib was then determined following SDS PAGE and autoradiography. The two right-hand lanes show lack of phosphorylation of hScrib in the absence of PKA, whilst the lower panels show the Coomassie stain of the gel demonstrating equal levels of the GST-hScrib fusion protein throughout. The quantitation of hScrib phosphorylation from three independent experiments is also shown.</p

Flowcytometric analysis of cell cycle in cancer cells treated with either NVP-BEZ235 or RAD001.

<p>(A–D) Cells (5×10⁵) were seeded in the presence of 10% serum and treated with NVP-BEZ235 or RAD001 for 48 h at a dose of 10 nM or 100 nM, respectively. A higher dose of NVP-BEZ235 (100 nM) significantly augmented the percentage of cells in the G0-G1phase of the cell cycle, compared with that of RAD001 (100 nM). (A)–(B); The data from two group A cells. (C)–(D); the data from two group B cells.</p

Copy number gain at the locus of

<p><b><i>K-Ras</i></b><b>(12p12.1) in the two group D cell lines.</b> (A) SNP array ‘karyograms’ (250K) of HEC-50B cells. The graph shows the total copy number through chromosome 9p-X. The locus of <i>K-Ras</i> is amplified as indicated. (B) Array CGH of KLE cells. The graphs show total copy number throughout the entire genome (Left) and chromosome 12 (Right). The locus of <i>K-Ras</i> is amplified as indicated.</p