Depletion of the Adaptor Protein NCK Increases UV-Induced p53 Phosphorylation and Promotes Apoptosis

<div><p>The cellular response to DNA damage requires the coordination of many proteins involved in diverse molecular processes. Discrete molecular pathways are becoming increasingly well understood, but the interconnectivity and coordination of multiple pathways remains less clear. We now show that NCK, an adapter protein involved in cytoskeletal responses to tyrosine kinase receptor signaling, accumulates in the nucleus in response to DNA damage and this translocation can be blocked by specific inhibition of the ATR protein kinase. Strikingly, HeLa cells depleted of NCK undergo apoptosis shortly after UV irradiation, as monitored by caspase-3 cleavage and PARP cleavage. This rapid, hyperactive apoptosis in NCK depleted cells might be p53 dependent, because loss of NCK also increased UV-induced p53 phosphorylation. Importantly, depletion of SOCS7, which is necessary for NCK nuclear translocation, phenocopies NCK depletion, indicating the nuclear accumulation of NCK is responsible for these molecular events. There are two NCK isoforms that have mostly redundant functions, and although NCK2 appears to have a greater contribution, depletion of NCK1 or NCK2, led to increased p53 phosphorylation and early apoptosis after UV exposure. These data reveal a novel function for NCK in regulating p53 phosphorylation and apoptosis, and provide evidence for interconnectedness of growth factor signaling proteins and the DNA damage response.</p> </div

Loss of SOCS7 prevents nuclear accumulation of NCK and results in early UV-induced apoptosis and elevated p53 phosphorylation.

<p>(A) HeLa cells transfected with control or SOCS7 siRNA were treated with 50 J/m² UV and allowed to recover for 1 hr before being fixed and stained with the indicated antibodies and DRAQ5. Scale bars are 20 µm. All images are confocal sections. Bar graphs below are ratio of nuclear to cytoplasmic fluorescence of NCK with siControl, no treat, defined as 1. n = 43-67; error bars represent SE; (**) P < 0.0001. (B) HeLa cells were treated as in A, and equal amounts of lysates were immunoblotted for cleaved CASP3, total CASP3, cleaved PARP, total PARP, and NCK. RAN was used as a loading control. n = 3. (C) RT-PCR of SOCS7 mRNA from HeLa cells transfected with control or SOCS7 siRNA. GAPDH was used as an internal control. RT = reverse transcriptase. n = 3. (D) HeLa cells transfected with control, NCK1 and NCK2, or SOCS7 siRNA were treated with 50 J/m² UV and allowed to recover for 2 hr before lysates were prepared. Equal amounts of lysates were immunoblotted for p53-pS15 (phospho-specific), CHK2-pT68 (phospho-specific), and NCK. RAN was used as a loading control. Band intensities were measured with ImageJ. Bar graph is ratio of p53-pS15 band intensity to RAN band intensity with siControl, no treat (NT), defined as 1. n = 4; error bars represent SE; (*) P < 0.005. (E) RT-PCR of p53 mRNA from HeLa cells transfected with control, NCK1 and NCK2, or SOCS7 siRNA. GAPDH was used as an internal control. Bar graph is ratio of p53 mRNA to GAPDH mRNA with siControl ratio defined as 1. n = 4; error bars represent SE. (F) 293T cells transfected with control, NCK1 and NCK2, or SOCS7 siRNA were treated with 50 J/m² UV and allowed to recover for 2 hr before lysates were prepared. Equal amounts of lysates were immunoblotted for p53-pS15, p53 (total protein), and NCK. RAN was used as a loading control. n = 3. (G) RT-PCR of SOCS7 mRNA from 293T cells transfected with control or SOCS7 siRNA. GAPDH was used as an internal control. n = 3.</p

DNA damage induces the nuclear accumulation of NCK.

<p>(A) HeLa cells were treated with 50 J/m² UV, 10 µM Etoposide, or 10 Gy IR and allowed to recover for 2 hr, 1 hr, and 1 hr, respectively, before being fixed and stained with the indicated antibodies and DRAQ5 to visualize nuclei. Scale bars are 20 µm. All images are confocal sections. (B) HeLa cells transfected with GFP-NCK1 and GFP-NCK2 were treated with 50 J/m² UV and allowed to recover for 2 hr before being fixed and stained with indicated antibodies and DRAQ5. Bar graphs below each panel are ratio of nuclear to cytoplasmic fluorescence of endogenous NCK, A, or GFP-NCK, B, with no treat, or vehicle, defined as 1. n = 19-82; error bars represent SE; (*) P < 0.0001.</p

Specific contributions of the NCK isoforms.

<p>(A) HeLa cells transfected with GFP-NCK1 or GFP-NCK2 were treated with 50 J/m² UV and allowed to recover for 2 hr before being fixed and stained with indicated antibodies and DRAQ5. Scale bars are 20 µm. All images are confocal sections. Bar graphs below each panel are ratio of nuclear to cytoplasmic fluorescence of GFP-NCK with no treat defined as 1. n = 14-28; error bars represent SE; (*) P < 0.001, (**) P < 0.0001. (B) HeLa cells transfected with control, NCK1, or NCK2 siRNA were treated with 50 J/m² UV and allowed to recover for 2 hr before lysates were prepared. Equal amounts of lysates were immunoblotted for p53-pS15 (phospho-specific) and NCK. RAN was used as a loading control. n = 3. (C) RT-PCR of p53 mRNA from HeLa cells transfected with control, NCK1, or NCK2 siRNA. GAPDH was used as an internal control. RT = reverse transcriptase. n = 4. (D) HeLa cells transfected with control, NCK1, or NCK2 siRNA were treated with 50 J/m² UV and allowed to recover for 2 hr before lysates were prepared. Equal amounts of lysates were immunoblotted for cleaved CASP3, total CASP3, cleaved PARP, total PARP, and NCK. RAN was used as a loading control. n = 3. (E) RT-PCR of NCK1 and NCK2 mRNA from HeLa cells transfected with control, NCK1, or NCK2 siRNA. GAPDH was used as an internal control. n = 3. (F) Cell lysates from HeLa cells transfected with control, NCK1, or NCK2 siRNA were immunoprecipitated with a NCK antibody, which detects NCK 1 and NCK2, or IgG control, and immunoblotted for NCK or NCK2. n = 4.</p

Loss of NCK causes early UV-induced apoptosis in HeLa cells.

<p>(A) HeLa cells transfected with control or NCK1 and NCK2 siRNA were treated with 50 J/m² UV and allowed to recover for 2 hr before being fixed and stained with the indicated antibody and DRAQ5. Scale bars are 20 µm. All images are confocal sections. section n = 3. (B) Cells were treated as in A, and equal amounts of lysates were immunoblotted for cleaved CASP3, total CASP3, cleaved PARP, total PARP, and NCK. RAN was used as a loading control. n = 4. (C) HeLa cells transfected with control or NCK1 and NCK2 siRNA were treated with 50 J/m² UV and allowed to recover for the indicated amount of time before cell viability was assayed. Percent cell viability for each siRNA at time 0 hr (no treat), is defined as 100%. n = 8; error bars represent SE; (*) P < 0.05, (**) P < 0.01, (#) P < 0.001, (# #) P < 0.0001.</p

ATR activity is necessary for nuclear accumulation of NCK.

<p>(A) HeLa cells were pretreated for 30 min with 50 µM wortmannin and then treated with 50 J/m² UV and allowed to recover for 2 hr in the presence of wortmannin before being fixed and stained with the indicated antibodies and DRAQ5. Scale bars are 20 µm. All images are confocal sections. Bar graph below is ratio of nuclear to cytoplasmic fluorescence of NCK with vehicle pretreated, no treat, defined as 1. n = 125-174; error bars represent SE; (*) P < 0.0001. (B) Same as in A, except 5 µM ATR inhibitor (VE-821) was used, and n = 79-117.</p

The Scribble Polarity Protein Stabilizes E-Cadherin/p120-Catenin Binding and Blocks Retrieval of E-Cadherin to the Golgi

<div><p>Several polarity proteins, including Scribble (Scrb) have been implicated in control of vesicle traffic, and in particular the endocytosis of E-cadherin, but through unknown mechanisms. We now show that depletion of Scrb enhances endocytosis of E-cadherin by weakening the E-cadherin-p120catenin interaction. Unexpectedly, however, the internalized E-cadherin is not degraded but accumulates in the Golgi apparatus. Silencing p120-catenin causes degradation of E-cadherin in lysosomes, but degradation is blocked by the co-depletion of Scrb, which diverts the internalized E-cadherin to the Golgi. Loss of Scrb also enhances E-cadherin binding to retromer components, and retromer is required for Golgi accumulation of Scrb, and E-cadherin stability. These data identify a novel and unanticipated function for Scrb in blocking retromer-mediated diversion of E-cadherin to the Golgi. They provide evidence that polarity proteins can modify the intracellular itinerary for endocytosed membrane proteins.</p> </div

Internalized E-cadherin is retrieve to the Golgi in Scrb-depleted cells, is associated with retromer components, and requires retromer for Golgi accumulation.

<p>(A) Staining of Ecad-GFP expressing MDCK cells for mannose-6 phosphate receptor. Scale bars are 20 µm. (B) Co-localization of Ecad-GFP with the Golgi marker GM130. All images are confocal sections (Zeiss LSM 510; 40× oil immersion lens, NA 1.4). Overlap coefficients were determined using Openlab software (+/−1 SEM; n = 3). (C) Co-localization of Ecad-GFP with Golgi 58K. Overlap coefficients were determined using Openlab software (+/−1 SEM; n = 3).</p

The E-cadherin/p120 interaction is disrupted in Scrb-depleted cells.

<p>(A) E-cadherin was immunoprecipitated from control and ScrbKD lysates as described in Methods, and the samples were blotted for p120. Band intensities were measured as described in Methods. Error bars = mean +/−1 SEM (n = 4). (B) Equal amounts of LucKD and ScrbKD total cell lysates were blotted for p120. Error bars indicate mean +/− SEM (n = 3). (C) β-catenin was immunoprecipitated from control and ScrbKD MDCK cell lysates and the samples were blotted for E-cadherin (mouse antibody) and β-catenin (rabbit antibody). Error bars indicate mean +/− SEM (n = 3). (D) Control and Scrb-depleted cells stably expressing Ecad-GFP were grown on slides for 48 h, then fixed and immunostained for p120 or β-catenin. Scale bars are 20 µm. Pixel intensities were measured across the lines shown and are displayed beneath the images. Quantification of co-localization between Ecad-GFP and the catenins was performed for multiple fields using Openlab. Error bars show mean +/−SEM (n = 5 fields per condition).</p

E-cadherin endocytosis is promoted in the absence of Scrb.

<p>(A) Schematic of experimental protocol to distinguish effects of Scrb depletion on delivery of E-cadherin to the plasma membrane versus on internalization of E-cadherin from the membrane. The rr1 antibody, which recognizes the extracellular domain of E-cadherin, will be retained at the plasma membrane at 4°C. Internalization is slow in control cells, so most of the antibody will still be at the plasma membrane after 2 h at 37°C. However, if internalization is accelerated by loss of Scrb, the rr1 antibody will be recruited along with E-cadherin into intracellular vesicles. (B) Control and Scrb-depleted Ecad-GFP cells were plated on 0.4 µm filters for 18 h. The E-cadherin extracellular domain-specific antibody (rr1) was added to the bottom wells and allowed to bind to cells for 1 h at 4°C. Cells were then washed to remove unbound antibody, incubated at either 4°C or 37°C for 2 h, then fixed and stained for the antibody (red) and for Scrb (blue). All images are confocal sections (Zeiss LSM 510; 40× oil immersion lens, NA 1.4). Scale bars are 20 µm. (C) Quantification of the overlap coefficient for colocalization of Ecad-GFP and rr1 (mean +/−1 SEM; n = 5 fields).</p