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

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