Structure-Function Analysis of Nucleolin and ErbB Receptors Interactions

The ErbB receptor tyrosine kinases and nucleolin are major contributors to malignant transformation. Recently we have found that cell-surface ErbB receptors interact with nucleolin via their cytoplasmic tail. Overexpression of ErbB1 and nucleolin leads to receptor phosphorylation, dimerization and anchorage independent growth.In the present study we explored the regions of nucleolin and ErbB responsible for their interaction. Using mutational analyses, we addressed the structure–function relationship of the interaction between ErbB1 and nucleolin. We identified the ErbB1 nuclear localization domain as nucleolin interacting region. This region is important for nucleolin-associated receptor activation. Notably, though the tyrosine kinase domain is important for nucleolin-associated receptor activation, it is not involved in nucleolin/ErbB interactions. In addition, we demonstrated that the 212 c-terminal portion of nucleolin is imperative for the interaction with ErbB1 and ErbB4. This region of nucleolin is sufficient to induce ErbB1 dimerization, phosphorylation and growth in soft agar.The oncogenic potential of ErbB depends on receptor levels and activation. Nucleolin affects ErbB dimerization and activation leading to enhanced cell growth. The C-terminal region of nucleolin and the ErbB1 NLS-domain mediate this interaction. Moreover, when the C-terminal 212 amino acids region of nucleolin is expressed with ErbB1, it can enhance anchorage independent cell growth. Taken together these results offer new insight into the role of ErbB1 and nucleolin interaction in malignant cells

Nucleolin and ErbB1- induced anchorage-independent growth.

<p>(A) Rat-1 cells stably expressing empty vector (not shown), ErbB1, nucleolin or ErbB1 and nucleolin were seeded in soft agar (6000 cells/well in 96 well plates) in medium containing 10% FBS, 0.3% agar, in the presence of 100 ng/ml EGF. The extent colony formation was determined 2 weeks later. Cells were dyed with MTT and the wells were photographed and colonies were counted. (B) Results were quantified using image analyzer program Image pro-Plus. The results are presented as total number of colonies and size of colonies. Note that nucleolin+ErbB1 but not each protein alone induced more and larger colonies. Results are the mean±SD 6 determinations. Each experiment was repeated at least three times with 3 different clones, with similar results.</p

Nucleolin induces ErbB phosphorylation in a ligand independent manner.

<p>(A) COS7 cells were transiently co-transfected with expression vector of either ErbB1 or ErbB2 receptors alone or with Myc-Nucleolin. Following 30 min serum deprivation, cells were either untreated or treated with EGF 100 ng/ml for 5 min. Cell lysates were immunoblotted with anti-phosphorylated EGFR or anti-phosphorylated ErbB2 antibodies respectively. As control, lysates were immunoblotted with anti-EGFR or anti-ErbB2 antibodies. Note that at time 0 phosphorylated receptors are detected in cells expressing nucleolin and EGFR or ErbB2. (B) COS7 cells were transiently co-transfected with expression vector of each of the ErbB receptors and Myc-Nucleolin. Following 30 min serum deprivation cells were untreated or treated with either EGF 100 ng/ml or NRG 100 ng/ml for 5 min as indicated. Cell lysates were subjected to immunoprecipitation with anti-phosphotyrosine antibodies (PY20) and immunoblotted with specific ErbB antibodies. Note that in untreated cells, phosphorylated receptor is detected in cells expressing nucleolin and ErbB receptor. The values represent fold induction compared to the receptor levels in untreated cells (1).</p

Nucleolin binds to the four ErbB4 isoforms.

<p>(A) COS7 cells were transiently co-transfected with expression vector of ErbB4 and either Myc-Nucleolin, or Myc-TGFβIIR. Cell lysates were subjected to immunoprecipitation with anti-Myc antibodies and immunoblotted with anti-ErbB4 antibodies, as a control the total cell lysates were reacted with anti-ErbB4 antibodies. (B) COS7 cells were transiently co-transfected with or without expression vector of each of the ErbB4 receptor isoforms and Myc-GFP-Nucleolin. Cell lysates were subjected to immunoprecipitation with anti-ErbB4 antibodies. The immunoprecipitated proteins as well as total cell lysates were immunoblotted with anti-GFP antibodies or with anti Myc antibodies as indicated.</p

Nucleolin reduces ErbB1 disappearance.

<p>(A) Total cell lysates of Rat-1 cells stably expressing ErbB1, nucleolin or ErbB1 and nucleolin were immonoblotted with anti-Myc to recognize Myc-Nucleolin or anti-GFP to identify GFP-ErbB1. (B) Rat-1 cells stably expressing empty vector, ErbB1, nucleolin or ErbB1 and nucleolin were grown in 6 wells plates (10⁶ cells/well). Cells were deprived of serum for 24 h then treated with EGF 100 ng/ml for the indicated time periods. Total cell lysates were analyzed by Western blot, using anti-ErbB1 antibodies. As control total cell lysate was immunoblotted with anti-tubulin antibodies. Note that in the presence of nucleolin the EGFR is more stabilized following EGF stimulation.</p

Nucleolin binds to the four ErbB receptors.

<p>(A) COS7 cells were transiently co-transfected with expression vector of each of the ErbB receptors and Myc-Nucleolin. Cell lysates were separated into cytosolic (supernatant) and total membrane (pellet) fractions. The pellet fraction was subjected to immunoprecipitation with anti-Myc (nucleolin) or anti-HA (control) antibodies and immunoblotted with specific ErbBs antibodies. Total pellet and supernatant extracts are shown in the right lanes of each panel. (B) COS7 cells were transiently co-transfected with expression vector of ErbB1 and Myc-Nucleolin. Nuclear and non-nuclear cell extracts were immunoblotted with anti-ErbB1 antibodies. As control for the fractionation purity Blots were reacted with anti-PARP antibodies (as a nuclear marker) and anti-tubulin antibodies (as a cytosolic marker). (C) Cell lysates prepared from SKBR3 cells were subjected to immunoprecipitation with either anti-nucleolin or anti ErbB2 antibodies and immunoblotted with either anti- ErbB2 or anti-nucleolin antibodies as indicated, as a control total cell lysates are shown in the right lane of each panel.</p

Nucleolin binds the cytoplasmic tail of ErbB4.

<p>(A) SDS-PAGE of Coomassie blue–stained proteins isolated from PC12 cell extracts, loaded on GST-ErbB4 agarose affinity matrix or control GST agarose matrix. The arrow indicates a specific 110-kD band, which was identified as nucleolin by mass spectroscopy. (B) COS7 cells were transiently transfected with expression vector of Myc-Nucleolin. Cell lysates were incubated with immobilized GST-ErbB4 or GST. Proteins retained on the beads were resolved by SDS-PAGE and then processed for Western blot using anti-Myc antibodies. (C) Cell lysates prepared from Du145 cells were incubated with immobilized GST-ErbB4 or GST. Eluates from GST-ErbB4 and GST control affinity matrices were resolved by SDS-PAGE and then processed for Western blot using a monoclonal mouse anti-nucleolin antibody.</p

Nucleolin induces ErbB1 dimerization.

<p>COS7 cells were transiently co-transfected with expression vector of ErbB1 receptors alone or with Myc-Nucleolin. Following 30 min serum deprivation cells were untreated or treated with EGF 100 ng/ml for 5 min. Cell lysates were incubated with BS³ crosslinker (2 mM) for 1 h and immunoblotted with anti-EGFR antibodies. Note that in the presence of nucleolin receptor dimmers appears to be similar to the level of receptor dimmers induced by EGF in the absence of nucleolin.</p