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 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

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 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 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

Analysis of the ErbB1 regions important for ErbB1 nucleolin interaction.

<p>(A) Schematic presentation of ErbB1 deletion mutants. (B) Cells were co-transfected with pEGFP-nucleolin-Myc and the indicated ErbB1 mutant expression vectors. The resulting cell lysates were subjected to immunoprecipitation with anti-Myc antibodies and immunoblotted with monoclonal antibody directed to the extracellular portion of ErbB1 (anti-111). Total cell lysates are shown.</p

The NLS of ErbB1 is important for ErbB1 nucleolin interaction.

<p>(A) Schematic presentation of the chimeric protein expression vectors. (B) Cells were transfected with nucleolin-myc and either pEGFP (empty vector), pEGFP-TM-NLS or pEGFP-TM expression vectors. The resulting cell lysates were subjected to fractionation and the pellets were subjected to immunoprecipitation with anti-Myc antibodies and immunoblotted with anti GFP antibodies. As control, total cell lysates were blotted with anti-GFP and anti-Myc antibodies.</p

Cellular distribution of ErbB1 and nucleolin mutants.

<p>HEK-293T cells were transfected with EGFP-ErbB1 and either pDsRed, pDsRed -nucleolin, pDsRed -N-ter or pDsRed -212-C-ter. 48 h following transfection cells were fixed and subjected to confocal microscopy analysis. GFP-ErbB1 is visualized in green and pDsRed expression vectors are seen in red. Representative cells are presented.</p